Tumour necrosis factor binding ligands

ABSTRACT

The present invention relates to ligands which bind to human tumour necrosis factor alpha (TNF) in a manner such that upon binding of these ligands to TNF the biological activity of TNF is modified. In preferred forms the ligand binds to TNF in a manner such that the induction of fibrin deposition in the tumour and induction of endothelial procoagulant activity of the TNF is inhibited; the binding of TNF to receptors on endothelial cells, tumour regression, cytotoxicity and receptor binding activities of the TNF on tumour cells are unaffected. The ligand is preferably an antibody, F(ab) fragment, single domain antibody (dABs), single chain antibody or a serum binding protein. It is preferred, however, that the ligand is a monoclonal antibody or F(ab) fragment thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/326,996(now U.S. Pat. No. 7,517,963), filed Jan. 5, 2006, which is acontinuation of application Ser. No. 10/702,681, filed Nov. 5, 2003 (nowabandoned), which is a continuation of application Ser. No. 10/453,176,filed Jun. 2, 2003 (now abandoned), which is a continuation ofapplication Ser. No. 10/359,934, filed Feb. 7, 2003 (now abandoned),which is a continuation of application Ser. No. 10/327,541, filed Dec.20, 2002 (now abandoned), which is a continuation of application Ser.No. 10/265,451 (now abandoned), filed Oct. 3, 2002, which is acontinuation of application Ser. No. 09/736,630 (now U.S. Pat. No.6,593,458), filed Dec. 13, 2000, which is a continuation of applicationSer. No. 09/364,039 (now U.S. Pat. No. 6,416,757), filed Jul. 30, 1999,which is a continuation of application Ser. No. 08/823,893, filed Mar.17, 1997 (now U.S. Pat. No. 5,959,087), which is a continuation ofapplication Ser. No. 08/344,133, filed Nov. 23, 1994 (now U.S. Pat. No.5,644,034), which is a continuation-in-part of application Ser. No.07/828,956, filed Feb. 18, 1992 (now abandoned), which is a nationalphase filing of international application PCT/AU90/00337, filed 7 Aug.,1990, published in English on Feb. 21, 1991, which claims the benefit ofAustralian applications AU PJ5662, filed Aug. 7, 1989, and AU PJ7576,filed Nov. 24, 1989, the disclosures of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to ligands which bind to human tumournecrosis factor alpha (TNF) in a manner such that upon binding thebiological activity of TNF is modified. The type of modification shownhere is distinct from previous descriptions of antibodies which bind toTNF alpha and inhibit all TNF alpha activity. The new discovery showshow the different activities of TNF alpha can be selectively inhibitedor enhanced. In addition, the present invention relates to a compositioncomprising a molecule bound to TNF and to methods of therapy utilisingTNF and molecules active against TNF.

BACKGROUND OF THE INVENTION

Tumor necrosis factor alpha (TNF) is a product of activated macrophagesfirst observed in the serum of experimental animals presensitized withBacillus Calmette-Guerin or Corynebacterium parvum and challenged withendotoxin (LPS). Following the systematic administration of TNFhaemorrhagic necrosis was observed in some transplantable tumours ofmice while in vitro TNF caused cytolytic or cytostatic effects on tumourcell lines.

In addition to its host-protective effect, TNF has been implicated asthe causative agent of pathological changes in septicemia, cachexia andcerebral malaria. Passive immunization of mice with a polyclonal rabbitserum against TNF has been shown to protect mice against the lethaleffects of LPS endotoxin, the initiating agent of toxic shock, whenadministered prior to infection.

The gene encoding TNF has been cloned allowing the usefulness of thismonokine as a potential cancer therapy agent to be assessed. While THFinfusion into cancer patients in stage 1 clinical trials has resulted intumour regression, side-effects such as thrombocytopaenia,lymphocytopaenia, hepatotoxicity, renal impairment and hypertension havealso been reported. These quite significant side-effects associated withthe clinical use of TNF are predictable in view of the many knowneffects of TNF, some of which are listed in Table 1.

TABLE 1 BIOLOGICAL ACTIVITIES OF TNF ANTI-TUMOUR ANTI-VIRALANTI-PARASITE FUNCTION cytotoxic action on tumour cells pyrogenicactivity angiogenic activity inhibition of lipoprotein lipase activationof neutrophils osteoclast activation induction of endothelial, monocyteand tumour cell procoagulant activity induction of surface antigens onendothelial cells induction of IL-6 induction of c-myc and c-fosinduction of EGF-receptor induction of IL-1 induction of TNF synthesisinduction of GM-CSF synthesis increased prostaglandin and collagenasesynthesis Induction of acute phase protein C3

Of particular importance is the activation of coagulation which occursas a consequence of TNF activation of endothelium and also peripheralblood monocytes. Disseminated intravascular coagulation is associatedwith toxic shock and many cancers including gastro-intestinal cancer,cancer of the pancreas, prostate, lung, breast and ovary, melanoma,acute leukaemia, myeloma, myeloproliferative syndrome and myeloblasticleukaemia. Clearly modifications of TNF activity such that tumourregression activity remains intact but other undesirable effects such asactivation of coagulation are removed or masked would lead to a moreadvantageous cancer therapy, while complete abrogation of TNF activityis sought for successful treatment of toxic shock.

Segregation of hormonal activity through the use of site-specificantibodies (both polyclonal and monoclonal) can result in enhancedhormonal activity (Aston et al, 1989, Mol. Immunol. 26, 435). To datefew attempts have been made to assign antigenicity or function toparticular regions of the TNF molecule for which the three-dimensionalstructure is now known. Assignment of function to such regions wouldpermit the development of MAbs and other ligands of therapeutic use.Polyclonal antibodies to amino acids 1 to 15 have been reported to blockHela R19 cell receptor binding by TNF (Socher et al, 1987, PNAS, 84,8829) whilst monoclonal antibodies recognizing undefined conformationalepitopes is on TNF have been shown to inhibit TNF cytotoxicity in vitro(Bringman and Aggarwal, 1987, Hybridoma 6, 489). However, the effects ofthese antibodies on other TNF activities is unknown.

SUMMARY OF THE PRESENT INVENTION

The present inventors have produced panels of monoclonal antibodiesactive against human TNF and have characterized them with respect totheir effects on the anti-tumour effect of TNF (both in vitro and invivo), TNF receptor binding, activation of coagulation (both in vitroand in vivo) and defined their topographic specificities. This approachhas led the inventors to show that different topographic regions of TNFalpha are associated with different activities. Therefore the inventorsenable the identification of antibodies or ligands which selectivelyenhance or inhibit TNF alpha activity, thereby providing for improvedtherapeutic agents and regimes including TNF alpha.

In a first aspect the present invention consists in a ligand capable ofbinding to human TNF, the ligand being characterized in that when itbinds to TNF the following biological activities of the TNF areinhibited: —

1. Tumour regression;

2. Induction of endothelial procoagulant;

3. Induction of tumour fibrin deposition;

4. Cytotoxicity; and

5. Receptor binding.

In a preferred embodiment of all aspects the present invention theligand is selected from the group consisting of antibodies, F(ab)fragments, restructured antibodies (CDR grafted humanised antibodies)single domain antibodies (dAbs), single chain antibodies, serum bindingproteins, receptors and natural inhibitors. The ligand may also be aprotein or peptide which has been synthesised and which is analogous toone of the foregoing fragments. However, it is presently preferred thatthe ligand is a monoclonal antibody or F(ab) fragment thereof.

In a second aspect the present invention consists in a ligand capable ofbinding to human TNF, the ligand being characterized in that when itbinds to TNF the induction of endothelial procoagulant, tumourregression, induction of tumour fibrin deposition, cytotoxicity andreceptor binding activities of the TNF are inhibited, the ligand bindingto the TNF such that the epitope of the TNF defined by the topographicregions of residues 1-18(Val₁-Arg₂-Ser₃-Ser₄-Ser₅-Arg₆-Thr₇-Pro₈-Ser₉-Asp₁₀-Lys₁₁-Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈),58-65 (Ile₅₈-Tyr₅₉-Ser₆₀-Gln₆₁-Val₆₂-Leu₆₃-Phe₆₄-Lys₆₅), 115-125(Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅)and 138-149(Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉),or the topographic region of residues 1-18(Val₁-Arg₂-Ser₃-Ser₄-Ser₅-Arg₆-Thr₇-Pro₈-Ser₉-Asp₁₀-Lys₁₁-Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈),108-128(Gly₁₀₈-Ala₁₀₉-Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇-Lys₁₂₈),or the topographic region of residues 56-79(Tyr₅₆-Leu₅₇-Ile₅₈-Tyr₅₉-Ser₆₀-Gln₆₁-Val₆₂-Leu₆₃-Phe₆₄-Lys₆₅-Gly₆₆-Gln₆₇-Gly₆₈-Cys₆₉-Pro₇₀-Ser₇₁-Thr₇₂-His₇₃-Val₇₄-Leu₇₅-Thr₇₇-His₇₈-Thr₇₉).110-127(Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇)and 135-155(Glu₁₃₅-Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃-Ile₁₅₄-Ile₁₅₅)is substantially prevented from binding to naturally occurringbiologically active ligands.

In a third aspect the present invention consists in a ligand which bindsto human TNF in at least two regions selected from the group consistingpredominantly of the topographic region of residues 1-20(Val₁-Arg₂-Ser₃-Ser₄-Ser₅-Arg₆-Thr₇-Pro₈-Ser₉-Asp₁₀-Lys₁₁-Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈-Asn₁₉-Pro₂₀),the topographic region of residues 56-77(Tyr₅₆-Leu₅₇-Ile₅₈-Tyr₅₉-Ser₆₀-Gln₆₁-Val₆₂-Leu₆₃-Phe₆₄-Lys₆₅-Gly₆₆-Gln₆₇-Gly₆₈-Cys₆₉-Pro₇₀-Ser₇₁-Thr₇₂-His₇₃-Val₇₄-Leu₇₅-Leu₇₆-Thr₇₇),the topographic region of residues 108-127(Gly₁₀₈-Ala₁₀₉-Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇)and the topographic region of residues 138-149(Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉).

In a preferred embodiment of the third aspect of the present inventionthe ligand binds to human TNF in the topographic regions of residues1-18, 58-65, 115-125 and 138-149. Such sequence regions aretopographically represented in FIG. 23.

In a further preferred embodiment of the third aspect of the presentinvention the ligand binds to human TNF in the topographic regions ofresidues 1-18(Val₁-Arg₂-Ser₃-Ser₅-Arg₆-Thr₇-Pro₈-Ser₉-Asp₁₀-Lys₁₁-Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈)and 108-128(Gly₁₀₈-Ala₁₀₉-Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇-Lys₁₂₈).Such sequence regions are topographically represented in FIG. 24.

In a further preferred embodiment of the second aspect of the presentinvention the ligand binds to human TNF in the topographic regions ofresidues 56-79, 110-127 and 136-155. Such sequence regions aretopographically represented in FIG. 25.

In a particularly preferred embodiment of the first, second and thirdaspects of the present invention the ligand is a monoclonal antibodyselected from the group consisting of the monoclonal antibodiesdesignated MAb 1, MAb 47 and MAb 54. Samples of the hybridoma cell lineswhich produce MAb 1, MAb 54 and MAb 47 have been deposited with theEuropean Collection of Animal Cell Cultures (ECACC), Vaccine Researchand Production Laboratory, Public Health Laboratory Service, Centre forApplied Microbiology and Research, Porton Down, Salisbury, Wiltshire 5P4OJG, United Kingdom. MAb 1 was deposited on 3 Aug. 1989 and accordedAccession No. 89080301; MAb 54 was deposited on 31 Aug. 1989 andaccorded Accession No. 89083103; MAb 47 was deposited on 14 Dec. 1989and accorded Accession No. 89121402, under the terms and conditions ofthe Budapest Treaty for the Deposit of Microorganisms for Patentpurposes.

In a fourth aspect the present invention consists in a compositioncomprising TNF in combination with the ligand of the first, second orthird aspect of the present invention, characterised in that the ligandis bound to the TNF.

In a fifth aspect the present invention consists in a method of treatingtoxic shock comprising administering either the ligand of the first,second or third aspect of the present invention or the composition ofthe fourth aspect of the present invention.

In a sixth aspect the present invention consists in a ligand capable ofbinding to human TNF, the ligand being characterised in that when itbinds to TNF the induction of endothelial procoagulant activity of theTNF is inhibited; binding of TNF to receptors on endothelial cells isinhibited; the induction of tumour fibrin deposition and tumourregression activities of the TNF are enhanced; the cytotoxicity isunaffected and tumour receptor binding activities of the TNF areunaffected or enhanced.

In a seventh aspect the present invention consists in a ligand capableof binding to human TNF, the ligand being characterized in that when itbinds to TNF the induction of endothelial procoagulant activity of theTNF is inhibited; the binding of the TNF to receptors on endothelialcells is inhibited, the induction of tumour fibrin deposition and tumourregression activities of the TNF are enhanced; and the cytotoxicity andreceptor binding activities of the TNF are unaffected; the ligandbinding to the TNF such that the epitope of the TNF defined by thetopographic regions of residues 1-30, 117-128 and 141-153 issubstantially prevented from binding to naturally occurring biologicallyactive ligands.

In an eighth aspect the present invention consists of a ligand whichbinds to human TNF in the topographic regions of residues 1-30, 117-128and 141-153.

In a preferred embodiment of the eighth aspect of the present inventionthe ligand binds to human TNF in the topographic regions of residues1-26, 117-128 and 141-153. Such sequence regions are topographicallyrepresented in to FIG. 26.

In a preferred embodiment of the sixth, seventh and eighth aspects ofthe present invention the ligand is the monoclonal antibody designatedMAb 32. A sample of the hybridoma producing MAb 32 was deposited withThe European Collection of Animal Cell Cultures (ECACC), VaccineResearch and Production Laboratory, Public Health Laboratory Service,Centre for Applied Microbiology and Research, Porton Down, Salisbury,Wiltshire 5P4 OJG, United Kingdom on 3 Aug. 1989 and was accordedAccession No. 89080302, under the terms and conditions of the BudapestTreaty for the Deposit of Microorganisms for Patent purposes.

In a ninth aspect the present invention consists in a compositioncomprising TNF in combination with a ligand of the sixth, seventh oreighth aspects of the present invention characterised in that the ligandis bound to TNF. No previous documentation of administering MAbs withTNF in order to modify activity of the administered cytokine exists.

In a tenth aspect the present invention consists in a method of treatingtumours the growth of which is inhibited by TNF, comprisingadministering either the ligand of the sixth, seventh or eighth aspectsof the present invention or the composition of the ninth aspect of thepresent invention.

In an eleventh aspect the present invention consists in a ligand whichbinds to residues 1-18 of human TNF (peptide 301).

In a twelfth aspect the present invention consists in a ligand capableof binding to human TNF, the ligand being characterized in that when itbinds to TNF the induction of endothelial procoagulant activity of theTNF is inhibited; the binding of TNF to receptors on endothelial cellsis inhibited; the induction of tumour fibrin deposition and tumourregression activities of the TNF are enhanced; the cytotoxicity of theTNF are unaffected and tumour receptor binding activities of the TNF areunaffected or enhanced, the ligand binding to TNF such that the epitopeof the TNF defined by the topographic region of residues 1-18 issubstantially prevented from binding to naturally occurring biologicallyactive ligands.

In a thirteenth aspect the present invention consists in a compositioncomprising TNF in combination with a ligand of the eleventh or twelfthaspects of the present invention characterized in that the ligand isbound to the TNF.

In a fourteenth aspect the present invention consists in a method oftreating tumours the growth of which is inhibited by TNF, comprisingadministering either the ligand of the eleventh or twelfth aspect of thepresent invention or the composition of the thirteenth aspect of thepresent invention.

In a fifteenth aspect the present invention consists in ligand capableof binding to human TNF, the ligand being characterised in that when itbinds to TNF the cytotoxicity and tumour regression activities of theTNF are unaffected; the induction of endothelial procoagulant andinduction of tumour fibrin deposition activities of the TNF areinhibited and receptor binding activities of the TNF are unaffected.

In a sixteenth aspect the present invention consists in a ligand capableof binding to human TNF, the ligand being characterized in that when itbinds to TNF the cytotoxicity and tumour regression activities of theTNF are unaffected; the induction of endothelial procoagulant andinduction of tumour fibrin deposition activities of the TNF areinhibited and the tumour receptor binding activities of the TNF areunaffected, the ligand binding to TNF such that the epitope of the TNFdefined by the topographic regions of residues 22-40(Ala₂₂-Glu₂₃-Gly₂₄-Gln₂₅-Leu₂₆-Gln₂₇-Trp₂₈-Leu₂₉-Asn₃₀-Arg₃₁-Arg₃₂-Ala₃₃-Asn₃₄-Ala₃₅-Leu₃₆-Leu₃₇-Ala₃₈-Asp₃₉-Gly₄₀),49-96(Val₄₉-Val₅₀-Pro₅₁-Ser₅₂-Glu₅₃-Gly₅₄-Leu₅₅-Tyr₅₆-Leu₅₇-Ile₅₈-Tyr₅₉-Ser₆₀-Gln₆₁-Val₆₂-Leu₆₃-Phe₆₄-Lys₆₅-Gly₆₆-Gln₆₇-Gly₆₈-Cys₆₉-Pro₇₀-Ser₇₁-Thr₇₂-His₇₃-Val₇₄-Leu₇₅-Leu₇₆-Thr₇₇-His₇₈-Thr₇₉-Ile₈₀-Ser₈₁-Arg₈₂-Ile₈₃-Ala₈₄-Val₈₅-Ser₈₆-Tyr₈₇-Gln₈₈-Thr₈₉-Lys₉₀-Val₉₁-Asn₉₂-Leu₉₃-Leu₉₄-Ser₉₅-Ala₉₆),110-127(Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇),and 136-153(Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃)is substantially prevented from binding to naturally occurringbiologically active ligands.

In a seventeenth aspect the present invention consists in a ligand whichbinds to human TNF in the topographic regions of residues 22-40(Ala₂₂-Glu₂₃-Gly₂₄-Gln₂₅-Leu₂₆-Gln₂₇-Trp₂₈-Leu₂₉-Asn₃₀-Arg₃₁-Arg₃₂-Ala₃₃-Asn₃₄-Ala₃₅-Leu₃₆-Leu₃₇-Ala₃₈-Asn₃₉-Gly₄₀),49-97(Val₄₉-Val₅₀-Pro₅₁-Ser₅₂-Glu₅₃-Gly₅₄-Leu₅₅-Tyr₅₆-Leu₅₇-Ile₅₈-Tyr₅₉-Ser₆₀-Gln₆₁-Val₆₂-Leu₆₃-Phe₆₄-Lys₆₅-Gly₆₆-Gln₆₇-Gly₆₈-Cys₆₉-Pro₇₀-Ser₇₁-Thr₇₂-His₇₃-Val₇₄-Leu₇₅-Leu₇₆-Thr₇₇-His₇₈-Thr₇₉-Ile₈₀-Ser₈₁-Arg₈₂-Ile₈₃-Ala₈₄-Val₈₅-Ser₈₆-Tyr₈₇-Gln₈₈-Thr₈₉-Lys₉₀-Val₉₁-Asn₉₂-Leu₉₃-Leu₉₄-Ser₉₅-Ala₉₆-Ile₉₇),110-127(Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇),and 136-153(Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃).Such sequence regions are topographically represented in FIG. 27.

In a preferred embodiment of the seventeenth aspect of the presentinvention the ligand binds to human TNF in the topographic regions ofresidues 22-40(Ala₂₂-Glu₂₃-Gly₂₄-Gln₂₅-Leu₂₆-Gln₂₇-Trp₂₈-Leu₂₉-Asn₃₀-Arg₃₁-Arg₃₂-Ala₃₃-Asn₃₄-Ala₃₅-Leu₃₆-Leu₃₇-Ala₃₈-Asn₃₉-Gly₄₀),49-96(Val₄₉-Val₅₀-Pro₅₁-Ser₅₂-Glu₅₃-Gly₅₄-Leu₅₅-Tyr₅₆-Leu₅₇-Ile₅₈-Tyr₅₉-Ser₆₀-Gln₆₁-Val₆₂-Leu₆₃-Phe₆₄-Lys₆₅-Gly₆₆-Gln₆₇-Gly₆₈-Cys₆₉-Pro₇₀-Ser₇₁-Thr₇₂-His₇₃-Val₇₄-Leu₇₅-Leu₇₆-Thr₇₇-His₇₈-Thr₇₉-Ile₈₀-Ser₈₁-Arg₈₂-Ile₈₃-Arg₈₄-Val₈₅-Ser₈₆-Tyr₈₇-Gln₈₈-Thr₈₉-Lys₉₀-Val₉₁-Asn₉₂-Leu₉₃-Leu₉₄-Ser₉₅-Ala₉₆),110-127(Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇),and 136-153(Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃).These regions being proximate in the 3D structure of TNF alpha.

In a preferred embodiment of the fifteenth, sixteenth and seventeenthaspects of the present invention the ligand is the monoclonal antibodydesignated MAb 42. A sample of the hybridoma cell line producing MAb 42was deposited with The European Collection of Animal Cell Cultures(ECACC), Vaccine Research and Production Laboratory, Public HealthLaboratory Service, Centre for Applied Microbiology and Research, PortonDown, Salisbury, Wiltshire SP4 OJG, United Kingdom on 3 Aug. 1989 andwas accorded Accession No. 89080304, under the terms and conditions ofthe Budapest Treaty for the Deposit of Microorganisms for Patentpurposes.

In an eighteenth aspect the present invention consists in a compositioncomprising TNF in combination with the ligand of the fifteenth,sixteenth or seventeenth aspects of the present invention, characterisedin that the ligand is bound to the TNF.

In a nineteenth aspect the present invention consists in a method oftreating tumours inhibited by the action of TNF comprising administeringthe ligand of the fifteenth, sixteenth or seventeenth aspects of thepresent invention or the composition of the eighteenth aspect of thepresent invention.

In a twentieth aspect the present invention consists in a ligand capableof binding to human TNF, the ligand being characterised in that when itbinds to TNF the tumour fibrin deposition activity of the TNF isenhanced; the induction of endothelial procoagulant activity of the TNFis unaffected and the cytotoxicity, tumour regression and receptorbinding activities of the TNF are inhibited.

In a twenty-first aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterized in thatwhen it binds to TNF the tumour fibrin deposition activity of the TNF isenhanced; the induction of endothelial procoagulant activity of the TNFis unaffected and the cytotoxicity, tumour regression and tumourreceptor binding activities of the TNF are inhibited, the ligand bindingto TNF such that the epitope of the TNF defined by the topographicregions of residues 12-22)(Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈-Asn₁₉-Pro₂₀-Gln₂₁-Ala₂₂),36-45 (Leu₃₆-Leu₃₇-Ala₃₈-Asn₃₉-Gly₄₀-Val₄₁-Glu₄₂-Leu₄₃-Arg₄₄-Asp₄₅),96-105(Ala₉₆-Ile₉₇-Lys₉₈-Ser₉₉-Pro₁₀₀-Cys₁₀₁-Gln₁₀₂-Arg₁₀₃-Glu₁₀₄-Thr₁₀₅) and132-157(Leu₁₃₂-Ser₁₃₃-Ala₁₃₄-Glu₁₃₅-Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃-Ile₁₅₄-Ile₁₅₅-Ala₁₅₆-Leu₁₅₇)is substantially prevented from binding to naturally occurringbiologically active ligands.

In a twenty-second aspect the present invention consists in a ligandwhich binds to human TNF in the topographic regions of residues 12-22(Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈-Asn₁₉-Pro₂₀-Gln₂₁-Ala₂₂),36-45 (Leu₃₆-Leu₃₇-Ala₃₈-Asn₃₉-Gly₄₀-Val₄₁-Glu₄₂-Leu₄₃-Arg₄₄-Asp₄₅),96-105(Ala₉₆-Ile₉₇-Lys₉₈-Ser₉₉-Pro₁₀₀-Cys₁₀₁-Gln₁₀₂-Arg₁₀₃-Glu₁₀₄-Thr₁₀₅) and132-157(Leu₁₃₂-Ser₁₃₃-Ala₁₃₄-Glu₁₃₅-Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃-Ile₁₅₄-Ile₁₅₅-Ala₁₅₆-Leu₁₅₇).These regions are proximate in the 3D structure of TND and aretopographically represented in FIG. 28.

In a preferred embodiment of the twentieth, twenty-first andtwenty-second aspects of the present invention the ligand is themonoclonal antibody designated MAb 25. A sample of the hybridoma cellline producing MAb 25 was deposited with the European Collection ofAnimal Cell Cultures (ECACC), Vaccine Research and ProductionLaboratory, Public Health Laboratory Service, Centre for AppliedMicrobiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG,United Kingdom on 14 Dec. 1989 and was accorded Accession No. 89121401,under the terms and conditions of the Budapest Treaty for the Deposit ofMicroorganisms for Patent purposes.

In a twenty-third aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to TNF the tumour fibrin deposition activity of the TNF isenhanced and the cytotoxicity, tumour regression, induction ofendothelial procoagulant and receptor binding activities of the TNF areinhibited.

In a twenty-fourth aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterized in thatwhen it binds to TNF the tumour fibrin deposition activity of the TNF isenhanced and the cytotoxicity, tumour regression, induction ofendothelial procoagulant and tumour receptor binding activities of theTNF are inhibited, the ligand binding to the TNF such that the epitopeof the TNF defined by the topographic regions of residues 1-20(Val₁-Arg₂-Ser₃-Ser₄-Ser₅-Arg₆-Thr₇-Pro₈-Ser₉-Asp₁₀-Lys₁₁-Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈-Asn₁₉-Pro₂₀)and 76-90(Leu₇₆-Thr₇₇-His₇₈-Thr₇₉-Ile₈₀-Ser₈₁-Arg₈₂-Ile₈₃-Ala₈₄-Val₈₅-Ser₈₆-Tyr₈₇-Gln₈₈-Thr₈₉-Lys₉₀)is substantially prevented from binding to naturally occurringbiologically active ligands.

In a twenty-fifth aspect the present invention consists in a ligandwhich binds to human TNF in the topographic regions of residues 1-20 and76-90. These regions are proximate in the 3D structure of TNF and aretopographically represented in FIG. 29.

In a preferred embodiment of the twenty-fifth aspect of the presentinvention the ligand binds to TNF in the topographic regions of residues1-18(Val₁-Arg₂-Ser₃-Ser₄-Ser₅-Arg₆-Thr₇-Pro₈-Ser₉-Asp₁₀-Lys₁₁-Pro₁₂-Val₁₃-Ala₁₄-His₁₅-Val₁₆-Val₁₇-Ala₁₈)and 76-90(Leu₇₆-Thr₇₇-His₇₈-Thr₇₉-Ile₈₀-Ser₈₁-Arg₈₂-Ile₈₃-Ala₈₄-Val₈₅-Ser₈₆-Tyr₈₇-Gln₈₈-Thr₈₉-Lys₉₀).

In a preferred embodiment of the twenty-third, twenty-fourth andtwenty-fifth aspects of the present invention the ligand is themonoclonal antibody designated MAb 21. A sample of the hybridoma cellline producing MAb 21 was deposited with the European Collection ofAnimal Cell Cultures (ECACC), Vaccine Research and ProductionLaboratory, Public Health Laboratory Service Centre for AppliedMicrobiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG,United Kingdom on 25 Jan. 1990 and was accorded Accession No. 90012432,under the terms and conditions of the Budapest Treaty for the Deposit ofMicroorganisms for Patent purposes.

In a twenty-sixth aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to TNF the fibrin deposition activity of the TNF isunaffected and the cytotoxicity, tumour regression, induction ofendothelial procoagulant and tumour receptor binding activities of theTNF are inhibited.

In a twenty-seventh aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterized in thatwhen it binds to TNF the tumour fibering deposition activity of the TNFis unaffected and the cytotoxicity, tumour regression, induction ofendothelial procoagulant and receptor binding activities of the TNF areinhibited, the ligand binding to the TNF such that the epitope of theTNF defined by the topographic regions of residues 22-40(Ala₂₂-Glu₂₃-Gly₂₄-Gln₂₅-Leu₂₆-Gln₂₇-Trp₂₈-Leu₂₉-Asn₃₀-Arg₃₁-Arg₃₂-Ala₃₃-Asn₃₄-Ala₃₅-Leu₃₆-Leu₃₇-Ala₃₈-Asn₃₉-Gly₄₀),69-97(Cys₆₉-Pro₇₀-Ser₇₁-Thr₇₂-His₇₃-Val₇₄-Leu₇₅-Leu₇₆-Thr₇₇-His₇₈-Thr₇₉-Ile₈₀-Ser₈₁-Arg₈₂-Ile₈₃-Ala₈₄-Val₈₅-Ser₈₆-Tyr₈₇-Gln₈₈-Thr₈₉-Lys₉₀-Val₉₁-Asn₉₂-Leu₉₃-Leu₉₄-Ser₉₅-Ala₉₆-Ile₉₇),105-128(Thr₁₀₅-Pro₁₀₆-Glu₁₀₇-Gly₁₀₈-Ala₁₀₉-Glu₁₁₀-Ala₁₁₁-Lys₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇-Lys₁₂₈)and 135-155(Glu₁₃₅-Ile₁₃₆-Asn₁₃₇-Arg₁₃₈-Pro₁₃₉-Asp₁₄₀-Tyr₁₄₁-Leu₁₄₂-Asp₁₄₃-Phe₁₄₄-Ala₁₄₅-Glu₁₄₆-Ser₁₄₇-Gly₁₄₈-Gln₁₄₉-Val₁₅₀-Tyr₁₅₁-Phe₁₅₂-Gly₁₅₃-Ile₁₅₄-Ile₁₅₅)is substantially prevented from binding to naturally occurringbiologically active ligands.

In a twenty-eighth aspect the present intention consists in a ligandwhich binds to human TNF in the topographic regions of residues 22-40,69-97, 105-128 and 135-155. These regions are proximate in the 3Dstructure of TNF and are topographically represented in FIG. 30.

In a preferred embodiment of the twenty-sixth, twenty-seventh andtwenty-eighth aspects of the present invention the ligand is themonoclonal antibody designated MAb 53. A sample of the hybridoma cellline producing MAb 53 was deposited with the European Collection ofAnimal Cell cultures (ECACC), Vaccine Research and ProductionLaboratory, Public Health Laboratory Service, Centre for AppliedMicrobiology and Research, Porton Down, Salisbury, Wiltshire 5P4 OJG,United Kingdom on 25 Jan. 1990 and was accorded Accession No. 90012433,under the terms and conditions of the Budapest Treaty for the Deposit ofMicroorganisms for Patent purposes.

In a twenty-ninth aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to the TNF tumour fibrin deposition, induction ofendothelial procoagulant, cytotoxicity, tumour regression and receptorbinding activities of the TNF are unaffected.

In a thirtieth aspect the present invention consists in a ligand capableof binding to human TNF, the ligand being characterised in that when itbinds to TNF the tumour fibrin deposition, induction of endothelialprocoagulant, cytotoxicity, tumour regression and receptor bindingactivities of the TNF are unaffected, the ligand binding to TNF suchthat the epitope of the TNF defined by the topographic regions ofresidues 22-31 and 146-157 is substantially prevented from binding tonaturally occurring biologically active ligands.

In a thirty-first aspect the present invention consists in a ligandwhich binds to human TNF in the topographic regions of residues 22-31and 146-157. These regions are proximate in the 3D structure of TNF andare typographically represented in FIG. 31.

In a preferred embodiment of the twenty-ninth, thirtieth andthirty-first-aspects of the present invention the ligand is themonoclonal antibody designated MAb 37. A sample of the hybridoma cellline producing MAb 37 was deposited with the European Collection ofAnimal Cell Cultures (ECACC), Vaccine Research and ProductionLaboratory, Public Health Laboratory Service, Centre for AppliedMicrobiology and Research, Porton Down, Salisbury, Wiltshire 5P4 OJG,United Kingdom on 3 Aug. 1989 and was accorded Accession No. 89080303,under the terms and conditions of the Budapest Treaty for the Deposit ofMicroorganisms for Patent purposes.

In a thirty-second aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to TNF the induction of endothelial procoagulant activityof the TNF is unaffected and the cytotoxicity, tumour regression, tumourfibrin deposition, and receptor binding activities of the TNF areinhibited.

In a thirty-third aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to TNF the induction of endothelial procoagulant activityof the TNF is unaffected and the cytotoxicity, tumour regression, tumourfibrin deposition and receptor binding activities of the TNF areinhibited, the ligand binding to the TNF such that the epitope of theTNF defined by the topographic regions of residues 22-40 and 49-98 issubstantially prevented from binding to naturally occurring biologicallyactive ligands.

In a thirty-fourth aspect the present invention consists in a ligandwhich binds to human TNF in at least one of the regions selected fromthe group consisting of the topographic region of residues 22-40, thetopographic region of residues 49-98 and the topographic region ofresidues 69-97.

In a preferred embodiment of the thirty-fourth aspect of the presentinvention the ligand binds to human TNF in the topographical region ofresidues 49-98. This region is topographically represented in FIG. 32.

In a further preferred embodiment of the thirty-fourth aspect of thepresent invention the ligand binds to human TNF in the topographicregions of residues 22-40 and 70-87. These regions are proximate in the3D structure of TNF and are topographically represented in FIG. 33.

In a preferred embodiment of the thirty-second, thirty-third andthirty-fourth aspects of the present invention the ligand is monoclonalantibody MAb 11 or MAb 12.

In a thirty-fifth aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to TNF the induction of endothelial procoagulant activityof the TNF is inhibited.

In a thirty-sixth aspect the present invention consists in a ligandcapable of binding to human TNF, the ligand being characterised in thatwhen it binds to TNF the induction of endothelial procoagulant activityof the TNF is inhibited, the ligand binding to TNF such that the epitopeof the TNF defined by the topographical region of residues 108-128(Gly₁₀₈-Ala₁₀₉-Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₇-Ile₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇-Lys₁₂₈)is prevented from binding to naturally occurring biologically activeligands.

In a thirty-seventh aspect the present invention consists in a ligandwhich binds to human TNF in the topographical region of residues 108-128(Gly₁₀₈-Ala₁₀₉-Glu₁₁₀-Ala₁₁₁-Lys₁₁₂-Pro₁₁₃-Trp₁₁₄-Tyr₁₁₅-Glu₁₁₆-Pro₁₁₈-Tyr₁₁₉-Leu₁₂₀-Gly₁₂₁-Gly₁₂₂-Val₁₂₃-Phe₁₂₄-Gln₁₂₅-Leu₁₂₆-Glu₁₂₇-Lys₁₂₈).

In a preferred embodiment of the thirty-fifth, thirty-sixth andthirty-seventh aspects of the present invention the ligand is selectedfrom the group consisting of monoclonal antibodies designated MAb 1, MAb32, MAb 42, MAb 47, MAb 53 and MAb 54.

The biological activities of TNF referred to herein by the terms “TumourRegression”, “Induction of Endothelial Procoagulant”, “Induction ofTumour Fibrin Deposition”, “Cytotoxicty” and “Receptor Binding” are tobe determined by the methods described below.

The term “single domain antibodies” as used herein is used to denotethose antibody fragments such as described in Ward et al (Nature, Vol.341, 1989, 544-546) as suggested by these authors.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following example and accompanying figures in which: —

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a titration assay with MAb 1 against TNF;

FIG. 2 shows TNF MAb 1 scatchard plot and affinity is determination;

FIG. 3 shows the effect of anti-TNF monoclonal antibodies 1 and 32 onTNF cytotoxicity in WEHI-164 cells;

FIG. 4 shows the effect of MAb 1 on TNF-induced regression of a Meth Asolid tumour;

FIG. 5 shows the effect of MAbs 1 and 25 on TNF-induced Meth A Ascitestumour regression;

FIG. 6 shows the effect of anti-TNF MAbs on induction of endothelialcell procoagulant activity by TNF;

FIGS. 7 a, 7 b and 7 c show incorporation of labeled fibrinogen intotumours of tumour-bearing mice and the effect of anti-TNF MAbs;

FIG. 8 is a schematic representation of epitopes on TNF;

FIG. 9 shows the effect of anti-TNF MAbs on TNF-induced regression ofWEHI-164 tumours;

FIGS. 10 a and 10 b show the enhancement of TNF regression activity byMAb 32 in two experiments;

FIGS. 11 a and 11 b show the enhancement of TNF-induced tumourregression by MAb 32-dose response at day 1 and day 2;

FIG. 12 shows binding of radio labelled TNF to receptors on bovineaortic endothelial cells;

FIG. 13 shows receptor binding studies of TNF complexed with MAb 32(—♦—), control antibody

 and MAb 47 (—▪—) on melanoma cell line MM418E;

FIG. 14 shows receptor binding studies of TNF complexed with MAb 32(—♦—), control antibody

 and MAb 47 (—▪—) on melanoma cell line IGR3;

FIG. 15 shows receptor binding studies of TNF complexed with MAb 32(—♦—) control antibody

 and MAb 47 (—▪—) on bladder carcinoma cell line 5637;

FIG. 16 shows receptor binding studies of TNF complexed with MAb 32(—♦—), control antibody

 and MAb 47 (—▪—) on breast carcinoma cell line MCF7;

FIG. 17 shows receptor binding studies of TNF complexed with MAb 32(—♦—), control antibody

 and MAb 47 (—▪—) on colon carcinoma cell line B10;

FIG. 18 shows the effect on TNF-mediated tumour regression in vivo byMAb 32

 control MAb (□) and MAb 47 (*);

FIG. 19 shows the effect on TNF-mediated tumour regression in vivo bycontrol MAb, MAb 32 and univalent FAb′ fragments of MAb 32;

FIG. 20 shows the effect on TNF induced tumour regression by control MAb(▪), MAb 32

 and peptide 301 antiserum

;

FIGS. 21 a, 21 b and 21 c show MAb 32 reactivity with overlappingpeptides of 10 AA length; and

FIG. 22 shows a schematic three dimensional representation of the TNFmolecule.

FIG. 23 shows topographically the region of residues 1-20, 56-77,108-127 and 138-149;

FIG. 24 shows topographically the region of residues 1-18 and 108-128;

FIG. 25 shows topographically the region of residues 56-79, 110-127 and136-155;

FIG. 26 shows topographically the region of residues 1-26, 117-128 and141-153;

FIG. 27 shows topographically the region of residues 22-40, 49-97,110-127 and 136-153;

FIG. 28 shows topographically the region of residues 12-22, 36-45,96-105 and 132-157;

FIG. 29 shows topographically the region of residues 1-20 and 76-90;

FIG. 30 shows topographically the region of residues 22-40, 69-97,105-128 and 135-155;

FIG. 31 shows topographically the region of residues 22-31 and 146-157;

FIG. 32 shows topographically the region of residues 49-98;

FIG. 33 shows topographically the region of residues 22-40 and 70-87;

FIG. 34 shows results of an ELISA using samples containing varyinglevels of TNF; and

FIG. 35 shows the effect of VHP3-VλA2 on anti-tumour activity of TNF.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Animals and Tumour Cell Lines

In all experiments BALB/C female mice aged 10-12 weeks obtained from theCSIRO animal facility were used. Meth A solid tumour and Meth A ascitestumour cell lines were obtained from the laboratory of Dr. Lloyd J. Old(Sloan Kettering Cancer Centre) and the WEHI-164 fibrosarcoma line wasobtained from Dr. Geeta Chauhdri (John Curtin School of MedicalResearch, Australian National University).

Fusions and Production of Hybridomas

Mice were immunised with 10 ug human recombinant TNF intra-peritoneallyin Freund's complete adjuvant. One month later 10 ug TNF in Freund'sincomplete adjuvant was administered. Six weeks later and four daysprior to fusion selected mice were boosted with 10 ug TNF in PBS. Spleencells from immune mice were fused with the myeloma Sp2/0 according tothe procedure of Rathjen and Underwood (1986, Mol. Immunol. 23, 441).Cell lines found to secrete anti-TNF antibodies by radioimmunoassay weresubcloned by limiting dilution on a feeder layer of mouse peritonealmacrophages. Antibody subclasses were determined by ELISA (Misotest,Commonwealth Serum Laboratories).

Radioimmunoassay

TNF was iodinated using lactoperoxidase according to standardprocedures. Culture supernatants from hybridomas (50 ul) were incubatedwith 125I TNF (20,000 cpm in 50 ul) overnight at 4° C. before theaddition of 100 ul Sac-Cel (donkey anti-mouse/rat immunoglobulins coatedcellulose, Wellcome Diagnostics) and incubated for a further 20 minutesat room temperature (20° C.). Following this incubation 1 ml of PBS wasadded and the tubes centrifuged at 2,500 rpm for 5 minutes. Thesupernatant was decanted and the pellet counted for bound radioactivity.

Antibody-Antibody Competition Assays

The comparative specificities of the monoclonal antibodies weredetermined in competition assays using either immobilized antigen (LACT)or antibody (PACT) (Aston and Ivanyi, 1985, Pharmac. Therapeut. 27,403).

PACT

Flexible microtitre trays were coated with monoclonal antibody (sodiumsulphate precipitated globulins from mouse ascites fluid, 100 microgramsper ml in sodium bicarbonate buffer, 0.05M, pH 9.6) overnight at 4° C.prior to blocking non-specific binding sites with 1% bovine serumalbumin in PBS (BSA/PBS). The binding of 125I TNF to immobilisedantibody was determined in the presence of varying concentrations of asecond anti-TNF monoclonal antibody. Antibody and TNF were addedsimultaneously and incubated for 24 hours prior to washing with PBS (4times) and counting wells for bound radioactivity. 100% binding wasdetermined in the absence of heterologous monoclonal antibody while 100%competition was determined in the presence of excess homologousmonoclonal antibody. All dilutions were prepared in BSA/PBS.

LACT

The binding of protein A purified, radiolabelled monoclonal antibodiesto TNF coated microtitre wells was determined in the presence of varyingconcentrations of a second monoclonal antibody. Microtitre plates werecoated with TNF (50 micrograms per ml) as described above. Quantities ofcompeting antibodies (50 microlitres) were pre-incubated on plates for 4hour at 4° C. prior to addition of 125I monoclonal antibody (30,000 cpm)for a further 24 hours. Binding of counts to wells was determined afterfour washes with PBS. 100% binding was determined in the absence ofcompeting antibody while 100% competition was determined in the presenceof excess unlabelled monoclonal antibody.

WEHI-164 Cytotoxicity Assay

Bioassay of recombinant TNF activity was performed according to Espevikand Nissen-Meyer (1986, J. Immunol. Methods 95, 99). The effect of themonoclonal antibody on TNF activity was determined by the addition ofthe monoclonal antibody to cell cultures at ABT90.

Tumour Regression Experiments

Modulation of TNF-induced tumour regression activity by monoclonalantibodies was assessed in three tumour models: the subcutaneous tumoursWEHI-164 and Meth A sarcoma and the ascitic Meth A tumour. Subcutaneoustumours were induced by the injection of approximately 5×10⁵ cells. Thisproduced tumours of between 10-15 mm approximately 14 days later. Micewere injected intra-peritoneally with human recombinant TNF (10micrograms) plus monoclonal antibody (200 microlitres ascites globulin)for four consecutive days. Control groups received injections of PBSalone or TNF plus monoclonal antibody against bovine growth hormone. Atthe commencement of each experiment tumour size was measured withcalipers in the case of solid tumours or tumour-bearing animals weighedin the case of ascites mice. These measurements were taken dailythroughout the course of the experiment.

Radio-Receptor Assays

WEHI-164 cells grown to confluency were scrape harvested and washed oncewith 1% BSA in Hank's balanced salt solution (HBSS, Gibco). 100 ul ofunlabelled TNF (1-10,000 ng/tube) or monoclonal antibody (10 folddilutions commencing 1 in 10 to 1 in 100,000 of ascitic globulin) wasadded to 50 ul 125I THF (50,000 cpm). WEHI cells were then added (200microlitres containing 2×10⁶ cells). This mixture was incubated in ashaking water bath at 37° C. for 3 hours. At the completion of thisincubation 1 ml of HBSS was added and the cells spun at 16,000 rpm for30 seconds. The supernatant was discarded and bound 125I TNF in the cellpellet counted. All dilutions were prepared in HBSS containing 1% BSA.

Procoagulant Induction by TNF on Endothelial Cells

Bovine aortic endothelial cells (passage 10) were grown in RPMI-1640containing 10% foetal calf serum (FCS), penicillin, streptomycin, and2-mercaptoethanol at 37° C. in 5% CO₂. For induction of procoagulantactivity by THF the cells were trypsinised and plated into 24-wellCostar trays according to the protocol of Bevilacqua et al., 1986 (PNAS83, 4533). TNF (0-500 units/culture) and monoclonal antibody (1 in 250dilution of ascitic globulin) was added after washing of the confluentcell monolayer with HBSS. After 4 hours the cells were scrape harvested,frozen and sonicated. Total cellular procoagulant activity wasdetermined by the recalcification time of normal donor platelet-poorplasma performed at 37° C., 100 microlitres of citrated platelet-poorplasma was added to 100 ul of cell lysate and 100 ul of calcium chloride(30 mM) and the time taken for clot formation recorded. In someexperiments tumour cell culture supernatant was added to endothelialcells treated with TNF and/or monoclonal antibody (final concentrationof 1 in 2).

Incorporation of 125I Fibrinogen into Tumors of Mice Treated with TNFand Monoclonal Antibody

In order to examine the effect of TNF and monoclonal antibodies onfibrin formation in vivo BALB/c mice were injected subcutaneously withWEHI-164 cells (10⁵ cells/animal). After 7-14 days, when tumours reacheda size of approximately 1 cm in diameter, animals were injectedintra-peritoneally with TNF (10 ug/animal) and 125I human fibrinogen(7.5 ug/animal, 122 uCi/mg Amersham) either alone or in the presence ofmonoclonal antibody to human TNF (200 ul/animal ascitic globulin).Monoclonal antibody against bovine growth hormone was used as controlmonoclonal antibody. Two hours after TNF infusion incorporation of 125Ifibrinogen into mouse tissue was determined by removing a piece oftissue, weighing it and counting the sample in a gamma counter.

In all 13 monoclonal antibodies reacting with human TNF were isolated.These monoclonal antibodies were designated MAb 1, MAb 11, MAb 12, MAb20, MAb 21, MAb 25, MAb 31, MAb 32, MAb 37, MAb 42, MAb 47, MAb 53 andMAb 54. The effect of these monoclonal antibodies on the bioactivity ofhuman TNF is set out in Table 2.

As can be seen from Table 2, whilst some monoclonal antibodies inhibitboth anti-tumour activity and activation of coagulation by human TNF(MAb 1, 47 and 54) not all antibodies which inhibit the anti-tumouractivity inhibit activation of coagulation either in vitro or in vivo(MAb 11, 12, 25 and 53). Indeed MAb 21 which inhibited tumour regressionenhanced the activation of coagulation in vivo.

TABLE 2 EFFECT OF MONOCLONAL ANTIBODIES ON TNF BIOACTIVITY MONOCLONALANTIBODY TNF BIOACTIVTTY 1 11 12 20 21 25 31 32 37 42 47 53 54Cytotoxicity − − − 0 − − 0 0 0 0 − − − Tumour Regression − − − 0 − − 0 +0 0 − − − Induction of − 0 0 − − 0 0 − 0 − − − − Procoagulant(Endothelial Fibrin Deposition − − − + + + + + 0 − − 0 − (tumour)Receptor Binding − − − 0 − − 0 +/0* 0 0 − − − (WEHI - 164) + Enhancement0 No effect − Inhibition *Depending on MAb concentration in the case ofWEHI-164 tumour cells and tumour type (see FIGS. 3, 13-17).

MAbs 1, 47 and 54, which have been shown in competition binding studiesto share an epitope on TNF, can be seen to have highly desirablecharacteristics in treatment of toxic shock and other conditions ofbacterial, viral and parasitic infection where TNF levels are highrequiring complete neutralisation of TNF. Other monoclonal antibodiessuch as MAb 32 are more appropriate as agents for coadministration withTNF during cancer therapy since they do not inhibit tumour regressionbut do inhibit activation of coagulation. This form of therapy isparticularly indicated in conjunction with cytotoxic drugs used incancer therapy which may potentiate activation of coagulation by TNF(e.g. vinblastin, acyclovir, IFN alpha, IL-2, actinomycin D, AZT,radiotherapy, adriamycin, mytomycin C, cytosine arabinoside,dounorubicin, cis-platin, vincristine, 5-fluorouracil, bleomycin,(Watanabe N et al 1988 Immunopharmacol. Immunotoxicol. 10 117-127) or indiseases where at certain stages TNF levels are low (e.g. AIDS) andwhere individuals may have AIDS associated cancer e.g. Kaposi sarcoma,non-Hodgkins lymphoma and squamous cell carcinoma.

Monoclonal antibody MAb 1 has been found to have the followingcharacteristics: —

1. Binds human recombinant TNF alpha, but not human lymphotoxin (TNFbeta) or human interferon. Similarly MAb 1 does not cross-react withrecombinant murine TNF (FIG. 1).

2. MAb 1 is of the immunoglobulin type IgG1, K with an apparent affinityof 4.4×10⁻⁹ moles/litre (FIG. 2).

3. MAb neutralises the cytotoxic effect of recombinant human TNF onWEHI-164 mouse fibrosarcoma cells in culture. One microgram of MAb 1neutralizes approximately 156.25 units of TNF in vitro (FIG. 3).

4. MAb 1 neutralises the tumour regression activity of TNF in thefollowing mouse tumour models in vivo; WEHI-164 subcutaneous solidtumour, the Meth A subcutaneous solid tumour and the Meth A ascitestumour (FIGS. 4, 5 and 9).

5. MAb1 prevents cerebral damage caused by human TNF in mice infectedwith malarial parasites.

6. In radioreceptor assays MAb 1 prevents binding of TNF to receptors onWEHI-164 cells (Table 3).

7. MAb 1 inhibits the induction of procoagulant activity (tissue factor)on cultured bovine aortic endothelial cells (FIG. 6).

8. MAb 1 reduces the uptake of 125I fibrinogen into tumours of micetreated with TNF (FIGS. 7 a-c).

9. MAb 1 competes for binding of 125I TNF and thus shares an overlappingepitope with the following monoclonal antibodies: 21, 25, 32, 47, 54 and37.

10. MAb 1 does not compete for binding of 125I TNF with the followingmonoclonal antibodies: 11, 12, 42, 53, 31 and 20 (FIG. 8).

TABLE 3 RADIORECEPTOR ASSAY: INHIBITION OF TNF BINDING TO WEHI-164 CELLSBY MAb 1 TREATMENT % SPECIFIC BINDING MAb 1 1/10 0 1/100 21 1/1,000 491/10,000 73 1/100,000 105 cold TNF(ng/tube) 10,000 0  5,000 0  1,000 0  500 10   100 11    10 64    1 108    0 100

MAb 32 is an IgG2b,K antibody with an affinity for human TNF alpha of8.77×10⁻⁹ moles/litre as determined by Scatchard analysis. Thismonoclonal antibody does not react with either human TNF beta(lymphotoxin) or mouse TNF alpha.

As shown in FIG. 3 MAb 32 does not inhibit TNF cytotoxicity in vitro asdetermined in the WEHI-164 assay.

Monoclonal antibody 32 variably enhances TNF-induced tumour regressionactivity against WEHI-164 fibrosarcoma tumours implanted subcutaneouslyinto BALB/c mice at a TNF dose of 10 μg/day (see FIGS. 10 a-b and 11a-b). This feature is not common to all monoclonal antibodies directedagainst TNF (FIG. 9) but resides within the binding site specificity ofMAb 32 (FIG. 8) which may allow greater receptor mediated uptake of TNFinto tumour cells (see Table 4).

TABLE 4 BINDING OF TNF TO RECEPTORS ON WEHI-164 CELLS IN THE PRESENCE OFMAb 32 % BINDING¹²⁵ I-TNF MAB DILUTION CONTROL MAB MAB 32 1/10 36 1411/100 74 88 1/1000 101 83 1/10,000 92 82 1/100,000 97 93

Enhancement of TNF activity by MAb 32 at lower doses of TNF is such thatat least tenfold less TNF is required to achieve the same degree oftumour regression (see FIGS. 11 and 18). The results for day 1, 2.5 ugand 1 ug TNF and day 2, 5 ug, 2.5 ug and 1 ug are statisticallysignificant in a t-test at p<0.01 level. This level of enhancement alsoincreases the survival rate of recipients since the lower dose of TNFused is not toxic. FIG. 19 shows that univalent Fab fragments of MAb32also cause enhancement of TNF-induced tumour regression in the samemanner as whole MAb 32 (see below).

MAb 32 inhibits the expression of clotting factors on endothelial cellsnormally induced by incubation of the cultured cells with TNF (see FIG.6). This response may be mediated by a previously unidentified TNFreceptor which is distinct to the receptor found on other cells.

Conversely, MAb 32 enhances the in vivo activation of coagulation withinthe tumour bed as shown by the incorporation of radiolabelled fibrinogen(FIGS. 7 a-c). This may be due to activation of monocytes/macrophageprocoagulant and may provide further insight into the mechanism ofTNF-induced tumour regression.

The results obtained with MAb 32 are shown in comparison to otheranti-TNF MAbs in Table 2.

The ability of MAb 32 and MAb 47 to inhibit the binding of TNF toendothelial cells was also assessed. Bovine aortic endothelial (BAE)cells (passage 11) were plated in 24-well culture dishes (Corning) whichhad been pre-coated with gelatin (0.2%) and grown to confluence inMcCoys 5A (modified) medium supplemented with 20% foetal calf serum. Forthe radio-receptor assay all dilutions (of cold TNF and MAbs) were madein this medium. The BAE cells were incubated for one hour in thepresence of either cold TNF (0 to 100 ng) or MAb (ascites globulinsdiluted 1/100 to 1/100,000) and iodinated TNF (50,000 cpm). At the endof this time the medium was withdrawn and the cells washed before beinglysed with 1M sodium hydroxide. The cell lysate was then counted forbound radioactive TNF. Specific binding of labelled TNF to the cells wasthen determined.

The results obtained in this assay with MAb 32, MAb 47 and a control MAbare set out in FIG. 12.

The results obtained in the clotting assay using BAE cells cultured inthe presence of TNF and anti-TNF MAb correlate with the results obtainedin the BAE radioreceptor assay i.e. MAbs which inhibit the induction ofclotting factors on the surface of endothelial cells (as shown by theincrease in clotting time compared to TNF alone) also inhibit thebinding of TNF to its receptor. This is exemplified by MAbs 32 and 47.

MAb 32, which does not inhibit TNF binding to WEHI-164 cells, doesinhibit binding of TNF to endothelial cells. This result providessupport for the hypothesis that distinct functional sites exist on theTNF molecule and that these sites interact with distinct receptorsubpopulations on different cell types. Thus ligands which bind todefined regions of TNF are able to modify the biological effects of TNFby limiting its binding to particular receptor subtypes.

As shown in FIG. 12 MAb 47 is a particularly potent inhibitor of TNFinteraction with endothelial cells, the percentage specific binding at adilution of 1/100 to 1/10,000 being effectively zero.

Receptor Binding Studies of Human TNF Complexed with MAB 32 on HumanCarcinoma Cell Lines In Vitro

MAb 32 has been shown to enhance the anti-tumour activity of human TNF.The mechanisms behind the enhancement may include restriction of TNFbinding to particular (tumour) receptor subtypes but not others(endothelial) with subsequent decrease in TNF toxicity to non-tumourcells. This mechanism does not require enhanced uptake of TNF by tumourcells in in vitro assays. In addition, MAb 32 also potentiates thebinding of human TNF directly to TNF receptors on certain humancarcinoma cell lines.

Materials and Methods

The following human carcinoma cell lines have been assayed for enhancedreceptor-mediated uptake of TNF in the presence of MAb 32; B10, CaCo, HT29, SKC01 (all colon carcinomas), 5637 (Bladder-carcinoma), MM418E(melanoma), IGR3 (melanoma), MCF 7 (breast carcinoma). The cells werepropagated in either RPMI-1640 (MM418E) DMEM (CaCo and IGR 3) or Iscovesmodified DMEM (B10, HT 29, SK01, S637, MCF 7) supplemented with 10%foetal calf serum, penicillin/streptomycin and L-glutamine. Receptorassays were performed as previously described for endothelial cellsexcept that the incubation time with iodinated TNF was extended to 3hours for all but the B10 cells for which the radiolabel was incubatedfor 1 hour.

Results

Enhanced TNF uptake was observed in the presence of MAb32 by themelanoma cell lines tested MM418E and IGR 3 (FIGS. 13 and 14), thebladder carcinoma 5637 (FIG. 15), and the breast carcinoma MCF 7 (FIG.16). MAb 32 did not affect TNF-receptor interaction in any of the othercell lines as shown by B 10 (FIG. 17) MAb 47, which has been shown toinhibit TNF binding to WEHI-164 cells and endothelial cells, and whichalso inhibits TNF-mediated tumour regression was found to markedlyinhibit TNF binding to all the cell lines tested (FIGS. 13-17).

Conclusions

Receptor binding analyses have indicated a second mechanism whereby MAb32 may potentiate the anti-tumour activity of TNF. This second pathwayfor enhancement of TNF results from increased uptake of TNF by tumourall receptors in the presence of MAb 32.

Enhancement of TNF-Mediated Tumour Regression In Vitro by MAB 32 orUnivalent FAB′ Fragments of MAB 32

Tumour regression studies were carried out as described above in micecarrying WEHI-164 subcutaneous tumours (N=5 animals/group). Tumour sizewas determined daily during the course of the experiment. The resultsobtained using MAb 32 are set out in FIG. 22 and show the mean+/−SD %change in tumour area at the completion of treatment (day 2) (▪ MAb 32:

 control MAb: *MAb 47). Differences observed between control MAb-TNF andMAb 32-TNF treated groups are statistically significant in a T-test atthe p-<0.01 level.

The results using the univalent FAb′ fragments of MAb 32 are shown inFIG. 19. Tumour size was determined daily during the course of theexperiment. The results show the mean SD % change in tumour area at thecompletion of treatment (day 2). Differences between the control MAb-10Fand MAb 32-TNF treated groups are statistically significant in a T-testat the P-<0.01 level.

TNF Induced Tumour Regression: Effect of Anti-Peptide 301 SERA

FIG. 20 shows the percent change in tumour area in tumour-bearing micetreated for three days with TNF plus control MAb (antibody againstbovine growth hormone), TNF plus MAb 32 or TNF plus antiserum (globulinfraction) against peptide 301. In an unpaired T-test the control groupis significantly different from both of the test groups (MAb 32,antiserum 301) while the MAb 32 and peptide antiserum 301 groups are notsignificantly different from each other. (control vs MAb 32, p<0.002;control vs antipeptide 301, p<0.025). Thus antisera raised using apeptide which comprises part of the MAb 32 specificity, also causes TNFenhancement of tumour regression.

As shown in FIG. 8 competition binding studies have shown that thethirteen monoclonal antibodies can be sub-divided into two main groups,namely MAbs 1, 21, 47, 54, 37, 32 and 25 and MAbs 11, 12, 53 and 42.Experiments were then conducted to identify the regions on human TNFrecognised by these monoclonal antibodies.

Identification of Regions on Human TNF Recognized by MonoclonalAntibodies

Methods

1. Overlapping peptides of 7 and 10 amino acid residues long weresynthesized on polypropylene pins according to the method of Geysen etal., 1984, PNAS 81, 3998-4002. The overlap was of 6 and 9 residuesrespectively and collectively the peptides covered the entire TNF aminoacid sequence. The peptides were tested for reactivity with the MAbs byELISA. MAbs which had TNF reactivity absorbed from them by priorincubation with whole TNF were also tested for reactivity with thepeptides and acted as a negative control.

2. Longer peptides of TNF were synthesized as described below. Thesepeptides were used to raise antisera in sheep using the followingprotocol. Merino sheep were primed with TNF peptide conjugated toovalbumin and emulsified in Freunds Complete adjuvant and boosted at 4weekly intervals with peptide-ovalbumin and sera assayed for thepresence of anti-TNF antibody by radioimmunoassay. Of the peptides shownonly peptides 275, 301, 305, 306 and 307 elicited sera reacting withwhole TNF. The positive sera were then used in competitive bindingassays (PACT assays) with the MAbs.

The following peptides were synthesised and are described using theconventional three letter code for each amino acid with the TNF sequenceregion indicated in brackets.

Peptide 275 H-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-OH (111-120)Peptide 301 H-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala-OH (1-18) Peptide 302H-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly- Leu-Tyr-Leu-Ile-OH(43-58) Peptide 304 H-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-OH (63-83) Peptide 305H-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-Glu-Ser-Gly-Gln-Val-OH (132-150) Peptide 306H-Val-Als-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly- Gln-Leu-OH (13-26)Peptide 307 H-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Ala-Asn-Gly-OH (22-40) Peptide 308H-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys- Gly-Gln-Gly-OH(54-68) Peptide 309 H-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-IIe-Ala-Val-Ser-Thr-Gln-Thr-Lys-Val-Asn-Leu-Leu-COOH (73-94) Peptide 323H-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Thr-Gln-Thr-OH (79-89)

Peptide 309

H-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Try-Gln-Thr-Lys-Val-Asn-Leu-Leu-COOH(73-94).

Peptide 323

H-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Tyr-Gln-Thr-COOH (79-89).

These peptides were synthesised using the following general protocol.

All peptide were synthesised using the Fmoc-polyamide method of solidphase peptide synthesis (Atherton et al, 1978, J. Chem. Soc. Chem.Commun., 13, 537-539). The solid resin used was PepSyn KA which is apolydimethylacrylamide gel on Kieselguhr support with4-hydroxymethylphenoxy-acetic acid as the functionalised linker(Atherton et al., 1975, J. Am. Chem. Soc. 97, 6584-6585).

The carboxy terminal amino acid was attached to the solid support by aDCC/DMAP-mediated symmetrical-anhydride esterification.

All Fmoc-groups were removed by piperidine/DMF wash and peptide bondswere formed either via pentafluorophenyl active esters or directly byBOP/NMM/HOBt (Castro's reagent) (Fournier et al, 1989, Int. J. PeptideProtein Res., 33, 133-139) except for certain amino acids as specifiedin Table 5.

Side chain protection chosen for the amino acids was removedconcomitantly during cleavage with the exception of Acm on cysteinewhich was left on after synthesis.

TABLE 5 Amino Acid Protecting Group Coupling Method Arg Mtr or PmcEither Asp OBut Either Cys Acm (permanent) Either Glu OBut Either HisBoc OPfp only Lys Boc Either Ser But BOP only Thr But BOP only Tyr ButEither Trp none Either Asn none OPfp only Gln none OPfp onlyCleavage and Purification

Peptide 301, 302, 305 are cleaved from the resin with 95% TFA and 5%thioanisole (1.5 h) and purified on reverse phase C4 column, (BufferA-0.1% aqueous TFA, Buffer B 80% CAN 20% A).

Peptide 303, 304 are cleaved from the resin with 95% TFA and 5% phenol(5-6 h) and purified on reverse phase C4 Column. (Buffers as above).

Peptide 306, 308 are cleaved from the resin with 95% TFA and 5% water(1.5 h) and purified on reverse phase C4 column. (Buffers as above).

Peptide 309 Peptide was cleaved from the resin with 95% TFA and 5%thioanisole and purified on reverse phase C4 column. (Buffers as above).

Peptide 307 Peptide was cleaved from the resin with a mixture of 93%TFA, 3.1% Anisole, 2.97% Ethylmethylsulfide and 0.95% Ethanedithiol (3h) and purified on reverse phase C4 column. (Buffers as above).

Results

Typical results of MAb ELISA using the 7 and 10 mers are shown in FIGS.21 a-c. Together with the results of PACT assays using the sheepanti-peptide sera (shown in Table 6) the following regions of TNFcontain the binding sites of the anti-TNF MAbs.

MAb 1: residues 1-18, 58-65, 115-125, 138-149

MAb 11: residues 49-98

MAb 12: residues 22-40, 70-87

MAb 21: residues 1-18, 76-90

MAb 25: residues 12-22, 36-45, 96-105, 132-157

MAb 32: residues 1-26, 117-128, 141-153

MAb 37: residues 22-31, 146-157

MAb 42: residues 22-40, 49-96, 110-127, 136-153

MAb 47: residues 1-18, 108-128

MAb 53: residues 22-40, 69-97, 105-128, 135-155

MAb 54: residues 56-79, 110-127, 136-155

TABLE 6 COMPETITIVE BINDING OF TNF BY ANTI-TNF MONOCLONES IN THEPRESENCE OF ANTI PEPTIDE SERA MAB/PEPTIDE SERA 275 301 305 306 307 1 − +− − − 11 − +/− − − − 12 − + − − ++ 21 − ++ − − − 25 − + − − − 32 −++++ + + − 37 − + +/− − + 47 − + − − − 53 − + − − + 54 − + − − − 42− + + − + Note 1: − indicates no competition, + indicates slightcompetititon at high concentration of anti-peptide antisera (1/50), ++++indicates trong competition by anti-peptide sera equal to that of thehomologous MAb. Note 2: Only peptide which elicited sera recognisingwhole TNF were used in this assay.

Note 1: − indicates no competition, + indicates slight competition athigh concentration of anti-peptide antisera (1/50), ++++ indicatesstrong competition by anti-peptide sera equal to that of the homologousMAb.

As will be understood by persons skilled in this field the ligands ofthe present invention can be used in assays of biological fluids fordetecting the presence of and quantifying the concentration of TNF in asample. One means by which this may be achieved is by using the ligandsof the present invention in conventional ELISAs. Set out below is anexample of such an assay.

TNF ELISA REAGENTS CARBONATE COATING BUFFER, pH 9.6 Na₂CO₃ 1.6 g NaHCO₃2.9 g Add 800 mL dH₂O, pH to 9.6 then make to 1 L with dH₂O BLOCKINGBUFFER BSA 1 g PBS 100 mL Add BSA to PBS and allow to dissolve fullybefore using. Store at 4° C. WASH BUFFER (0.05% TWEEN/PBS) TWEEN 20 0.5g PBS 1 L Add TWEEN to PBS and mix thoroughly before use CITRATE BUFFERCitric Acid. 2.11 g in 50 mL Add solutions together 1H₂O dH₂O and adjustpH to 4.0-4.2 TriSodium 1.47 g in 50 mL Citrate 2H₂O dH₂O NB: Allincubations can be carried out at 4° C. overnight OR at room temperaturefor 2 hrs OR at 37° C. for 1 hr.

-   NB: All incubations can be carried out at 4° C. overnight OR at room    temperature for 2 hrs OR at 37° C. for 1 hr.    Method    -   Coat ELISA plates with equal proportions of MAb1, MAb32 and        MAb54 to human TNF in carbonate coating buffer. The total        immunoglobulin concentration should be 20 μg/mL and 100 μL is        added to each well. Cover plates and incubate.    -   Wash plates 3× with PBS/TWEEN.    -   Incubate plates with 250 μL/well blocking buffer    -   Wash plates 3× with PBS/TWEEN.    -   Add 100 μL sample or TNF standards, diluted in blocking buffer        where required, to plates, then cover and incubate.    -   Wash plates 3× with PBS/TWEEN.    -   Add 100 μL biotinylated antibody mix (equal proportions of        biotinylated monoclonal antibodies 11 & 42 to human TNF) at a        final concentration of 10 μg/mL in blocking buffer to each well,        cover and incubate.    -   Wash plates 3× with PBS/TWEEN.    -   Add 100 μL/well streptavidin-peroxidase (Amersham product no.        RPN 1231) at 1/2,000 in blocking buffer, then cover and        incubate.    -   Wash plates 3× with PBS/TWEEN.    -   Add 100 μL/well biotinylated anti-stretpavidin monoclonal        antibody (Jackson Immunoresearch) at 1/40,000 in blocking        buffer, cover and incubate.    -   Wash plates 3× with PBS/TWEEN.    -   Add 100 μL/well streptavidin-peroxidase at 1/2,000 in blocking        buffer, cover and incubate.    -   Wash plates 3× with PBS/TWEEN.    -   Add 100 μL/well peroxidase substrate (ABTS) at 1 mg/mL in        citrate buffer containing 0.3 μL/ml H₂O₂ and leave to incubate        at room temperature for up to 1 hour.-   NB: Substrate solution should be prepared immediately prior to use.    -   Read absorbance at 405 nm, and compare sample readings with TNF        standard curve to determine TNF levels.    -   Read absorbance at 405 nm, and compare sample readings with TNF        standard curve to determine TNF levels

Biotinylation of IgG

50 mM Bicarbonate Buffer, pH 8.5

Na₂CO₃ 1.6 g NaHCO₃ 2.9 g In 1 L dH2O, adjust pH with HCl0.1 Phosphate Buffer, pH 7.0Method

-   -   Prepare immunoglobulins by purifying on a protein A column, then        freeze-drying.    -   Reconstitute the immunoglobulins with 50 mM bicarbonate buffer        to a concentration of 20 mg/mL in a clean glass test tube.    -   Add 0.4 mg biotin per 20 mg Ig directly to the tube.    -   Place the test tube on ice and incubate for 2 hours.    -   Remove the unreacted biotin by centrifuging at 1000 g for 15-30        minutes in a Centricon-30 microconcentrator. Dilute the sample        in 0.1M phosphate buffer and repeat the centrifugation twice.    -   Make the sample up to the original volume with phosphate buffer,        add 0.1% NaN₃ and store at 4° C. until used.

The results obtained in such an assay using samples containing knownamounts of TNF is shown in FIG. 34.

As mentioned above the specific mouse monoclonal antibodies disclosed inthis application can be humanised if required. A number of methods ofobtaining humanised antibodies are set out in PCT/GB92/01755(WO93/06213). A humanised version of MAb32 designated VHP3-VλA2 wasproduced by the method disclosed in PCT/GB92/01755. Briefly, thisantibody was produced as follows:

1 Cloning and Display of the V Genes of MAb 32 on Phage

Cloning of the V-Genes of MAb32:

The genes of the mouse MAb32 antibody (IgG2b, Kappa) were rescued by PCRessentially as described (Clackson et al., 1991, in “PCR: A PracticalApproach” eds. M. J. McPherson et al. IRL Press, Oxford, pp 187-214)using the primers VH1BACk and VH1FOR2 for the VH gene and Vk2BACK andVK4FOR for the VL gene and the polymerase chain reaction (PCR, R. K.Saiki et al., 1985, Science 230, p 1350). The mouse VH and Vk genes wereassembled for expression as scFv fragments by PCR assembly (Clackson etal., supra) amplified with VH1BACKSfi and VFFOR4NOT and ligated intophagemid pHEN1 (H. R. Hoogenboom et al., 1991 Nucl. Acids. Res. 19 pp4133-4137) as a SfiI-NotI cut restriction fragment, and electroporatedinto E. coli HB2 151 cells. Of 96 clones analysed by ELISA (see below),9 secreted TNF-binding soluble scFv fragments. Sequencing revealed inall clones a mouse VH of family IIB and a mouse Vk of family VI (E. A.Kabat et al., 1991 Sequences of Proteins of Immunological Interest, USPublic Health Services). Nucleotide mutations which were probablyintroduced by the PCR were detected by comparing the 9 sequences, and aclone with consensus sequence and binding activity (scFv-MAb32) chosenfor further cloning experiments.

Recloning of the MAb32 V-Genes for Soluble Expression:

The murine V-genes were recloned for soluble expression of heavy (Fd,VHCH1) or light chain, by linking the mouse V-genes to the human CR1 (ofthe mu-isotype) or human Ck gene respectively by splice overlapextension. The mouse Vk gene was amplified from scFv-MAb32 DNA witholigonucleotides MOJK1FORNX (binds in joining region of V-gene andMVKBASFI (binds in 5′ region and adds Sfil restriction site); the humanCk was obtained by PCR from a mouse-human chimaeric light chain gene (ofNQ10.12.5, described in Hoogenboom et al., 1991 supra), witholigonucleotides MOVK-HUCK-BACK (binds in 5′ of human Ck and ispartially complementary with mouse Jk 1 region) and HUCKNOT16NOMYC (sitsin 3′ end of human Ck, retains the terminal cysteine, and tags on a NotIrestriction site) as in Clackson et al., 1991 using a two fragmentassembly. For linkage of the DNA fragments, the two PCR fragments weremixed and amplified with MVKBASFI and HUCKNOT16NOMYC. The chimaeric VkCkgene was subsequently cloned as a SfiI-NotI fragment in pUC19 derivativecontaining the pe1B signal peptide sequence and appropriate cloningsites for soluble expression of the light chain (pUC19-pe1B-myc).Similarly, the mouse VH gene (amplified from scFv-MAb32 with LMB3 andVH1FOR-2) was combined by splicing by overlap extension PCR with thehuman u-CH1 domain (amplified from human IgM-derived cDNA (Marks et al.,1991, supra: WO 92/01047) with Mo-VH-Ku-CH1 and HCM1FONO, and cloned asSfiI-NotI fragment into a pUC19-pe1B-myc for soluble expression of atagged chain.

Display of the MAb32 Antibody on Phage:

The chimaeric light chain was displayed on phage fd by reamplificationof the mouse/human chimaeric chain with HUCKCYSNOT and MVKBAAPA andcloning into fd-tet-DOG1 as an ApaLI-NotI fragment. Cells harbouringplasmid with the heavy Fd chain gene were grown in 2×TY containingAMP-GLU (1%) to logarithmic phase (OD600 of 0-5) and infected with a20-fold excess of light-chain displaying phage. After 45 min at 37° C.without shaking and 45 min at 37° C. with shaking in the 2×TY,ampicillin (100 μg/ml). Glucose 1% medium, a sample was diluted into50-fold volume of prewarmed (37° C.) 2×TY, ampicillin (100 μg/ml) andtetracyclin (15 μg/ml), grown for 1 hr at 37° C. and then overnight at30° C. (shaking). Phage particles collected from the supernatant of suchculture displayed TNF-binding Fab fragments anchored through the lightchain on their surface.

Similarly, the reversed configuration was made. The heavy chain VHCH1fragment was cloned into fd-tet-DOG1 (after amplification of the Fdchain gene from the mouse/human chimeric construct with VH1BACKAPA andHCM1FONO), and phage used to infect cells capable of producing solublelight chain. Phage particles collected from the supernatant of suchculture displayed TNF-binding Fab fragments anchored through the heavychain VHCH1 fragment on their surface.

Properties of MAb 32 fragments displayed on phage:

The V-genes of the murine antibody MAb32 were cloned by amplifying thehybridoma V-genes, cloning the VH and Vk genes as scFv fragments inphagemid pHEN1 as above. Antibody scFv fragments which bind to TNF wereidentified by ELISA. The mouse VH gene was recloned in pUC19-pe1B-mycfor soluble expression as a mouse VH linked to human mu-CH1, while thelight chain was recloned with the human Ck domain in vector fd-tet-DOG1as a fusion with g3p. When cells harbouring the heavy chain constructwere infected with the fd-phage carrying the light chain, phageparticles emerged which carried light chain-g3p associated with the fdheavy chain. Indeed, binding to TNF and the 301 peptide was retained, asjudged by ELISA with phage displaying the mouse-human chimaeric Fabfragment. In the phage ELISA, the background signal of phage carryingthe light chain only was a lightly higher than wild-type fd-tet-DOG1phage, but always lower than the signal obtained with Fab-displayingphage. Similarly, TNF binding phage was made with the heavy chain VHCH1fragment anchored on phage, and the light chain provided as a solublefragment. Hence, MAb32 is functional in the dual combinatorial format inboth display orientations.

2 Chain Shuffling by Epitope Imprinted Selection (EIS) Construction ofOne Chain-Libraries:

Kappa, lambda light chain and Mu-specific cDNA was made from the mRNAprepared from the peripheral blood lymphocytes from two healthy donorsessentially as in Marks et al., 1991, supra. The first-strand cDNAsynthesis was performed with oligonucleotides HCM1FO, HUCLCYS andHUCKCYS for Mu-specific, lambda and kappa libraries respectively. TheVH-CH 1 repertoire was amplified from this cDNA with oligonucleotidesHCM1FO and six family specific VHBACK primers (as in Marks et al., 1991,supra), reamplified with a NotI-tagged forward primer (HCM1FONO) andApaLI tagged VHBACK primers (6 primers HuVH1BAAPA to HuVH6BAAPA).Similarly, the light chain repertoires were amplified with HUCLCYS orHUCKCYS forward primers and HUVλ1BACK to HuVλ6BACK or HuVk1BACK toHUVk6BACK back primers described in Marks et al., 1991, supra andPCT/GB91/01134 (WO 92/01047). In each case described in this section thelambda and kappa chain variable repertoires were amplified separately.The amplified repertoires were reamplified with ApaLI and NotI taggedversions of these oligonucleotides (13 back primers HuVλ1BAAPA toHuλ6BAAPA or HuVk1BAAPA to HuVkBAAPA and two forward priers HuCLCYSNOTand HuCKCYSNOT, respectively). All three repertoires were cloned intovector fd-tet-DOG1 as ApaLI-NotI fragments, and electroporated into E.coli MC1061 cells, to obtain libraries of 1.0×10⁷ clones for VλCA,1.4×10⁶ clones for VkCk, and 5×10⁶ clones for IgM-derived VHCH1. Thepresence of insert was checked and the frequency of inserts in thelibrary found to be higher than 95% in all three cases.

Selecting a Human VL Using the Mouse VH Domain as Docking Chain:

In a first chain shuffling experiment, the mouse VH (linked to the humanCH1 domain), expressed from pUC19-pelB-myc, was paired as Fab fragmentwith a library of 10⁷ different human VλCλ domains. Phage displaying theantibody fragments were subjected to rounds of panning on TNF-coatedtubes. By following the titre of the eluted phage, the extent ofselection was monitored. After 4 rounds (with a 100-fold increase in thetitre of eluted phage), 24 out of 28 individual clones were found to bebinding to TNF in an ELISA with phage expressing Fab fragments (all withthe mouse VH-human CH1). Phage only displaying the selected human VλCλdomains gave a background similar to phage displaying only the chimaericmouse Vk-human Ck. Sixteen clones taken after the first round ofselection were found to be negative.

Only three different BstN1 fingerprints were found amongst the 24binders, with one pattern dominating (21/24). Light chains VλA2, VλC4and VλD1 were found with frequencies of 21/24, 2/24 and 1/24respectively. Sequencing revealed that all three light chains arederived from the same germline gene, a human Vλ1-1-1. Clone VλC4 has 1,clone VλD1 has 2 and clone VλA2 7 amino-acid residue differences fromthe germline. However, clone VλA2 uses a framework-1 region which moreclosely resembled the germline sequence of a related Vλ1, humv1117, andtherefore may be the result of a cross-over. The germline character ofthe clones was also noted in the CDR3 sequence, with minimal variationin sequence and no length variation between the three clones.Apparently, only a very limited number of genes with very similarsequences fix the stringent requirements (being compatible with themouse VH and forming an antigen-binding pair).

Selecting a Human VH Using the Selected Human VL Domains as DockingChains:

Three selected Vλ genes were recloned in pUC19-pelB-myc for solubleexpression VλCλ chains. E. coli cells harbouring the three light chainplasmids were mixed, infected with a phage library of human VHCH1 genes,expressed from the fd-tet-DOC1 library described earlier and the librarysubjected to rounds of panning on TNF-coated Immuno tubes. Clones werepicked after 5 rounds, when the titre of eluted phage increased100-fold. Fifteen out of 20 clones analysed by BstNI fingerprint of theDNA insert used one of two patterns (with approximately the samefrequency). The 15 clones when combining their heavy chain VHCH1fragments with the VλA2 light chain gave stronger phase ELISA signalsthan when combined with the VλC4 or VλD1 light chain. Background signalsobtained with phage displaying the heavy chain VHCH1 fragment only weresimilar to the signal of the murine VH-human CH1.

Sequencing revealed that the two patterns could be assigned to threeunique human VH sequences (clones VHP1/2/3, with clone VHP1 having aBstNI fingerprint which is nearly identical to that of clone VHP2). Likethe selected light chain genes, the selected heavy chain genes arederived from the same germline VH gene (germline DP-51 from the VH3family, Tomlinson et al., J. Mol. Biol. 227, pp 776-798 1992), withminimal residue differences. The selected human V-genes were aligned totheir closest germline homologue; identical residues in the selectedgenes are represented by hyphens. Framework 4 of the V_(H) genes wastruncated at 4th residue. Clone VHP1 was most likely a cross-overbetween DP-51 and a related germline, DP-47. All three selected VH-geneshad relatively short CDR3 loops (8, 9 and 10 residues), but sharedlittle homology to this sequence.

Specificity of Binding of the Selected V-Gene Pairs:

A specificity ELISA with MAb32 and soluble ScFv fragments on a number ofantigens showed that MAb32, its ScFv-derivative and three of thehumanised TNF-binders (as ScFv-fragments) bind specifically to TNF. Nosignificant binding was obtained to ELISA plates coated with keyholelimpet haemocyanin, ovalbumin, cytochrome c. bovine serum albumin, humanthyroglobulin, or 2-phenyloxazol-5-one-BSA or to plastic only. Fullyhumanised clones were obtained which bound to both peptide 301 and TNF.

In addition, to show that the human scFv fragments compete with theoriginal antibody for binding to TNF, the binding of the scFv constructsin a competition ELISA with the Fab fragment derived by proteolyticcleavage of MAb32 was analysed. Single chain Fv fragments were incubatedon a TNF-coated surface with increasing amounts of the Fab fragment andthe amount of bound scFv detected in ELISA. Each of the scFv fragmentscompeted with the FabMAb32 for binding to TNF, including both theoriginal scFv-32 and the humanised scFv fragments.

Thus the fine specificity of MAb32 for peptide 301 of TNF was retainedthrough the humanisation process.

Affinity of Binding of the Selected V Gene Pairs:

MAb32 and purified, monomeric forms of the recombinant mouse scFv-MAb32and the human scFv antibodies VHP1-VλA2. VHP2-VλA2 and VHP3-VλA2, weresubjected to competition ELISA for the determination of the relativeaffinity for TNF. Antibodies were incubated on a TNF-coated surface inthe presence of increasing amounts of soluble TNF. All the clones showeda roughly similar decrease in the ELISA signal over the same range ofincreasing TNF concentrations (with an IC₅₀ in the 10 nM to 100 nMrange).

MAb32 and VHP3VλA2 fragments were also analysed for binding propertiesusing the Pharmacia BIAcore. TNF was indirectly immobilised on thesurface, and the binding of antibody monitored. On the TNF surface, theFab fragment from MAb32 by proteolytic cleavage and the scFv MAb32showed very similar fast off rates (approximately 10⁻² s⁻¹). The humanVHP3-VλA2 antibody has an off rate in the same range as the originalscFv-MAb32. On rates for antibody protein interactions were in the rangeseen for the interaction between other proteins and their receptors, andcover a 100 fold range between 10⁴ and 10⁶ M⁻¹ s⁻¹ (Mason D. W. andWilliams, A. F., 1986, Kinetics of Antibody Reactions and the Analysisof Cell Surface Antigens, Blackwell, Oxford; Pecht, I., 1992 in Sela, M.(ed), Dynamic Aspects of Antibody Function, Academic Press Inc., NewYork, Vol. 6 pp 1-68). Assuming the on rates of the antibody TNFinteractions are typical of antibody protein interactions, the off ratederived by the BIACore analysis is consistent with the affinityindicated by the competition ELISA (Kd≅10⁻⁷ to 10⁻⁸M).

Thus, these determinations are consistent with scFvMAb32 and thehumanised scFv clone VHP3-VλA2 having a similar affinity and thus withthe retention of affinity, as well as specificity, through epitopeimprinted selection.

Conclusion

We have shown that a mouse antibody can be rebuilt into a human antibodywith the same specificity by the process of epitope imprinted selection(EIS).

A library of human light chains were shuffled with a mouse VH domain,binding combinations selected and then used in a second shuffle as“docking domains” for a library of human VH genes. Completely humanantibodies were isolated from such “genuine” human library. Theantibodies were shown to bind retain binding specificity. Alternatively,the mouse VL was used as docking chain for selecting human VH partners.Such VH domains can be used to find human VL genes, or alternatively,can be combined with human VL domains selected with the mouse VH domain.Indeed, binding activity was obtained by combining two independentlyselected V-genes, pointing towards potential additivity of the EISprocedure.

The EIS approach may serve to humanise antibodies more rapidly than byCDR-grafting (Riechmann et al., 1988, supra), as this method requiresvery often a detailed knowledge of the 3-D structure of the antibody.However, the EIS method can be extended to for example antibodyrepertoires obtained by phage selection from immunised rodents.Following immunisation with antigen, a repertoire of V-genes with highaffinity and specificity may be selected and then used in an epitopeimprinted selection (see example 4) to generate a range of humanantibodies of high affinity and enriched for the desired specificity.

Enhancement of TNF-Induced Tumour Regression by Antibody VHP3-VλA2, theHuman Equivalent of MAB 32

BALB/c mice were inoculated with WEHI-164 tumour cells as describedabove. After development of subcutaneous tumours the mice were treateddaily with TNF (1 or 10 μg) alone or in combination with purified P3A2(50μ) by intraperitoneal injection. Tumour size was measured throughoutthe course of the treatment period.

Results are shown in FIG. 35.

VHP3-VλA2 enhanced the anti-tumour activity of TNF at both the 1 and 10μg levels.

CONCLUSIONS

Mapping of the regions recognised by each of the MAbs has indicated thatMAbs in group I (MAbs 1, 21, 47, 54, 37, 32 and 25) as shown on theschematic diagram bind TNF in the region of residues 1-18 with theexception of MAbs 37 and 54, while Mabs in group II of the schematicdiagram (MAbs 11, 12, 53 and 42) bind TNF in the region of residues70-96 which encompasses a so-called pallendromic loop on the TNF 3-Dstructure. Mabs which inhibit the induction of endothelial cellprocoagulant activity (MAbs 1, 32, 42, 47, 54 and 53) all bind in theregion of residues 108-128 which again contains a loop structure in the3-D model and may indicate that this region interacts with TNF receptorswhich are found on endothelial cells but not tumour cells. MAb 32 whichpotentiates the in vivo tumour regression and anti-viral activity of TNFis the only antibody which binds all the loop regions associated withresidues 1-26, 117-128, and 141-153 and hence binding of these regionsis crucial for enhanced TNF bioactivity with concomitant reduction oftoxicity for normal cells.

As is apparent from Table 2 MAb 1, 47 and 54 have the same effect on thebioactivity of TNF. From the results presented above it is noted thatthese three monoclonals bind to similar regions of the TNF molecule.Accordingly, it is believed that a ligand which binds to TNF in at leasttwo regions selected from the group consisting predominately of theregion of residues 1-20, the region of residues 56-77, the region ofresidues 108-128 and the region of residues 138-149 will effect thebioactivity of TNF in a manner similar to that of MAbs 1, 47 and 54.Similarly, it is believed that a ligand which binds to TNF predominatelyin the regions of residues 1-20 and 76-90 will have the same effect onthe bioactivity of TNF as MAb 21. A ligand which binds to TNFpredominately in the regions of residues 22-40 and 69-97 will have thesame effect on bioactivity of TNF as MAb 12. A ligand which binds to TNFpredominately in the regions of residues 1-30, 117-128, and 141-153would be expected to have the same effect on the bioactivity of TNF asMAb 32 and a ligand which binds to TNF predominately in the regions ofresidues 22-40, 49-97, 110-127 and 136-153 would be expected to have thesome effect on the bioactivity of TNF as MAb 42. A ligand which binds toTNF predominately in the regions of residues 22-31 and 146-157 would beexpected to have the same effect on the bioactivity of TNF as MAb 37 anda ligand which binds to TNF predominately in the regions of residues22-40, 69-97, 105-128 and 135-155 would be expected to have the sameeffect on the bioactivity of TNF as MAb 53.

The present inventors have quite clearly shown that the bioactivity ofTNF can be altered by the binding of a Ligand to the TNF, and that theeffect on the bioactivity is a function of the specificity of theligand. For example, the binding of MAb 32 to TNF in the regions ofresidues 1-26, 117-128 and 141-153 results in the induction ofendothelial procoagulant activity of the TNF and binding of TNF toreceptors on endothelial cells being inhibited; the induction of tumourfibrin deposition and tumour regression activities of the THF beingenhanced; the cytotoxicity being unaffected and the tumour receptorbinding activities of the TNF being unaffected or enhanced. It isbelieved that this effect on the bioactivity of the TNF may be due tothe prevention of the binding of the epitope of the TNF recognised byMAb 32 to naturally occurring biologically active ligands. Accordingly,it is believed that a similar effect to that produced by MAb 32 couldalso be produced by a ligand which binds to a region of TNF in a mannersuch that the epitope recognised by MAb 32 is prevented from binding tonaturally occurring biologically active ligands. This prevention ofbinding may be due to steric hindrance or other mechanisms.

Accordingly, it is intended that the prevention of the binding ofepitopes recognised by the various monoclonal antibodies describedherein to naturally occurring biologically active ligands is within thescope of the present invention.

1. An isolated antibody or fragment thereof that specifically binds tomature human TNF-α, wherein the antibody or fragment thereofcompetitively inhibits MAb 42 (ECACC Accession No. 89080304) forspecific binding to mature human TNF-α and, wherein when the antibody orfragment thereof binds mature human TNF-α, the induction of endothelialprocoagulant activity is inhibited, and the cytotoxicity, tumorregression and tumor receptor binding to TNF-α are not effected.
 2. Theantibody or fragment thereof according to claim 1, wherein the antibodyor fragment thereof competitively inhibits the binding of MAb 42 (ECACCAccession No. 89080304) to mature human TNF-α, where comparative bindingspecificity is determined by antibody-antibody competition assays in thepresence of mature human TNF-α.
 3. The antibody or fragment thereofaccording to claim 1 wherein the antibody is a monoclonal antibody. 4.The antibody or fragment thereof according to any one of claims 1-3,wherein the antibody is a humanized antibody.
 5. The antibody orfragment thereof according to any one of claims 1-3, wherein theantibody is a chimeric antibody.
 6. A composition comprising an isolatedantibody or fragment thereof that specifically binds to mature humanTNF-α, wherein the antibody or fragment thereof competitively inhibitsMAb 42 (ECACC Accession No. 89080304) for specific binding to maturehuman TNF-α and, wherein when the antibody or fragment thereof bindsmature human TNF-α, the induction of endothelial procoagulant activityis inhibited, and the cytotoxicity, tumor regression and tumor receptorbinding to TNF-α are not effected.
 7. The composition according to claim6, wherein the antibody or fragment thereof competitively inhibits thebinding of MAb 42 (ECACC Accession No. 89080304) to mature human TNF-α,where comparative binding specificity is determined by antibody-antibodycompetition assays in the presence of mature human TNF-α.
 8. Thecomposition according to claim 7 wherein the antibody is a monoclonalantibody.
 9. The composition according to any one of claims 6-8, whereinthe antibody is a humanized antibody.
 10. The composition according toany one of claims 6-8, wherein the antibody is a chimeric antibody. 11.An isolated single chain antibody that specifically binds to maturehuman TNF-α, wherein the antibody competitively inhibits MAb 42 (ECACCAccession No. 89080304) for specific binding to mature human TNF-α and,wherein when the antibody binds mature human TNF-α, the induction ofendothelial procoagulant activity is inhibited, and the cytotoxicity,tumor regression and tumor receptor binding to TNF-α are not effected.12. The antibody according to claim 11, wherein the antibodycompetitively inhibits the binding of MAb 42 (ECACC Accession No.89080304) to mature human TNF-α, where comparative binding specificityis determined by antibody-antibody competition assays in the presence ofmature human TNF-α.
 13. The antibody according to claim 11 or 12,wherein the antibody is a humanized antibody.
 14. The antibody accordingto claim 11 or 12, wherein the antibody is a chimeric antibody.
 15. Acomposition comprising an isolated single chain antibody thatspecifically binds to mature human TNF-α, wherein the antibodycompetitively inhibits MAb 42 (ECACC Accession No. 89080304) forspecific binding to mature human TNF-α and, wherein when the antibodybinds mature human TNF-α, the induction of endothelial procoagulantactivity is inhibited, and the cytotoxicity, tumor regression and tumorreceptor binding to TNF-α are not effected.
 16. The compositionaccording to claim 15, wherein the antibody competitively inhibits thebinding of MAb 42 (ECACC Accession No. 89080304) to mature human TNF-α,where comparative binding specificity is determined by antibody-antibodycompetition assays in the presence of mature human TNF-α.
 17. Thecomposition according to claim 15 or 16, wherein the antibody is ahumanized antibody.
 18. The composition according to claim 15 or 16,wherein the antibody is a chimeric antibody.
 19. An isolated singledomain antibody or fragment thereof that specifically binds to maturehuman TNF-α, wherein the antibody or fragment thereof competitivelyinhibits MAb 42 (ECACC Accession No. 89080304) for specific binding tomature human TNF-α and, wherein when the antibody or fragment thereofbinds mature human TNF-α, the induction of endothelial procoagulantactivity is inhibited, and the cytotoxicity, tumor regression and tumorreceptor binding to TNF-α are not effected.
 20. The antibody or fragmentthereof according to claim 19, wherein the antibody or fragment thereofcompetitively inhibits the binding of MAb 42 (ECACC Accession No.89080304) to mature human TNF-α, where comparative binding specificityis determined by antibody-antibody competition assays in the presence ofmature human TNF-α.
 21. The antibody or fragment thereof according toclaim 19 or 20, wherein the antibody is a humanized antibody.
 22. Theantibody or fragment thereof according to claim 19 or 20, wherein theantibody is a chimeric antibody.
 23. A composition comprising anisolated single domain antibody or fragment thereof that specificallybinds to mature human TNF-α, wherein the antibody or fragment thereofcompetitively inhibits MAb 42 (ECACC Accession No. 89080304) forspecific binding to mature human TNF-α and, wherein when the antibody orfragment thereof binds mature human TNF-α, the induction of endothelialprocoagulant activity is inhibited, and the cytotoxicity, tumorregression and tumor receptor binding to TNF-α are not effected.
 24. Thecomposition according to claim 23, wherein the antibody or fragmentthereof competitively inhibits the binding of MAb 42 (ECACC AccessionNo. 89080304) to mature human TNF-α, where comparative bindingspecificity is determined by antibody-antibody competition assays in thepresence of mature human TNF-α.
 25. The composition according to claim23 or 24, wherein the antibody is a humanized antibody.
 26. Thecomposition according to claim 23 or 24, wherein the antibody is achimeric antibody.
 27. An isolated antibody or fragment thereof thatspecifically binds to mature human TNF-α, wherein when the antibody orfragment thereof binds mature human TNF-α, the induction of endothelialprocoagulant activity is inhibited, wherein the antibody or fragmentthereof competitively inhibits a second antibody or fragment thereof forspecific binding to mature human TNF-α; and, wherein the second antibodyis MAb 42 (ECACC Accession No. 89080304).
 28. The antibody or fragmentthereof according to claim 27, wherein the antibody is a monoclonalantibody.
 29. The antibody or fragment thereof according to claim 27,wherein the antibody or fragment thereof inhibits the induction of tumorfibrin deposition activity.
 30. The antibody or fragment thereofaccording to claim 27, wherein the antibody or fragment thereof has noeffect on tumor receptor binding to TNF-α.
 31. The antibody or fragmentthereof according to claim 30, wherein the antibody or fragment thereofhas no effect on cytotoxicity.
 32. The antibody or fragment thereofaccording to claim 27, wherein the antibody or fragment thereof has noeffect on cytotoxicity.
 33. The antibody or fragment thereof accordingto claim 32, wherein the antibody or fragment thereof has no effect ontumor regression.
 34. The antibody or fragment thereof according toclaim 31, wherein the antibody or fragment thereof has no effect ontumor regression.
 35. The antibody or fragment thereof according toclaim 30, wherein the antibody or fragment thereof has no effect ontumor regression.
 36. The antibody or fragment thereof according toclaim 27, wherein the antibody or fragment thereof has no effect ontumor regression.
 37. The antibody or fragment thereof according toclaim 27, where comparative binding specificity between the antibody orfragment thereof and MAb 42 (ECACC Accession No. 89080304) is determinedby antibody-antibody competition assays in the presence of mature humanTNF-α.
 38. The antibody or fragment thereof according to claim 37,wherein the antibody or fragment thereof has no effect on tumor receptorbinding to TNF-α.
 39. The antibody or fragment thereof according toclaim 38, wherein the antibody or fragment thereof has no effect oncytotoxicity.
 40. The antibody or fragment thereof according to claim37, wherein the antibody or fragment thereof has no effect oncytotoxicity.
 41. The antibody or fragment thereof according to claim39, wherein the antibody or fragment thereof has no effect on tumorregression.
 42. The antibody or fragment thereof according to claim 37,wherein the antibody or fragment thereof has no effect on tumorregression.
 43. The antibody or fragment thereof according to any one ofclaims 27-42, wherein the antibody is a humanized antibody.
 44. Theantibody or fragment thereof according to any one of claims 27-42,wherein the antibody is a chimeric antibody.
 45. A compositioncomprising an isolated antibody or fragment thereof that specificallybinds to mature human TNF-α, wherein when the antibody or fragmentthereof binds mature human TNF-α, the induction of endothelialprocoagulant activity is inhibited, wherein the antibody or fragmentthereof competitively inhibits a second antibody or fragment thereof forspecific binding to mature human TNF-α, and, wherein the second antibodyis MAb 42 (ECACC Accession No. 89080304).
 46. The composition accordingto claim 45, wherein the antibody is a monoclonal antibody.
 47. Thecomposition according to claim 45, wherein the antibody or fragmentthereof inhibits the induction of tumor fibrin deposition activity. 48.The composition according to claim 45, wherein the antibody or fragmentthereof has no effect on tumor receptor binding to TNF-α.
 49. Thecomposition according to claim 48, wherein the antibody or fragmentthereof has no effect on cytotoxicity.
 50. The composition according toclaim 45, wherein the antibody or fragment thereof has no effect oncytotoxicity.
 51. The composition according to claim 50, wherein theantibody or fragment thereof has no effect on tumor regression.
 52. Thecomposition according to claim 49, wherein the antibody or fragmentthereof has no effect on tumor regression.
 53. The composition accordingto claim 48, wherein the antibody or fragment thereof has no effect ontumor regression.
 54. The composition according to claim 45, wherein theantibody or fragment thereof has no effect on tumor regression.
 55. Thecomposition according to claim 45, where comparative binding specificitybetween the antibody or fragment thereof and MAb 42 (ECACC Accession No.89080304) is determined by antibody-antibody competition assays in thepresence of mature human TNF-α.
 56. The composition according to claim55, wherein the antibody or fragment thereof has no effect on tumorreceptor binding to TNF-α.
 57. The composition according to claim 56,wherein the antibody or fragment thereof has no effect on cytotoxicity.58. The composition according to claim 55, wherein the antibody orfragment thereof has no effect on cytotoxicity.
 59. The compositionaccording to claim 57, wherein the antibody or fragment thereof has noeffect on tumor regression.
 60. The composition according to claim 57,wherein the antibody or fragment thereof has no effect on tumorregression.
 61. The composition according to any one of 52-60, whereinthe antibody is a humanized antibody.
 62. The composition according toany one of claims 52-60, wherein the antibody is a chimeric antibody.63. An isolated single chain antibody that specifically binds to maturehuman TNF-α, wherein when the antibody binds mature human TNF-α, theinduction of endothelial procoagulant activity is inhibited, wherein theantibody competitively inhibits a second antibody or fragment thereoffor specific binding to mature human TNF-α; and, wherein the secondantibody is MAb 42 (ECACC Accession No. 89080304).
 64. The antibodyaccording to claim 63, wherein the antibody inhibits the induction oftumor fibrin deposition activity.
 65. The antibody according to claim63, wherein the antibody has no effect on tumor receptor binding toTNF-α.
 66. The antibody according to claim 65, wherein the antibody hasno effect on cytotoxicity.
 67. The antibody according to claim 63,wherein the antibody has no effect on cytotoxicity.
 68. The antibodyaccording to claim 67, wherein the antibody has no effect on tumorregression.
 69. The antibody according to claim 66, wherein the antibodyhas no effect on tumor regression.
 70. The antibody according to claim65, wherein the antibody has no effect on tumor regression.
 71. Theantibody according to claim 63, wherein the antibody has no effect ontumor regression.
 72. The antibody according to claim 63, wherecomparative binding specificity between the antibody and MAb 42 (ECACCAccession No. 89080304) is determined by antibody-antibody competitionassays in the presence of mature human TNF-α.
 73. The antibody accordingto claim 72, wherein the antibody has no effect on tumor receptorbinding to TNF-α.
 74. The antibody according to claim 73, wherein theantibody has no effect on cytotoxicity.
 75. The antibody according toclaim 72, wherein the antibody has no effect on cytotoxicity.
 76. Theantibody according to claim 74, wherein the antibody has no effect ontumor regression.
 77. The antibody according to claim 72, wherein theantibody has no effect on tumor regression.
 78. The antibody accordingto any one of claims 63-77, wherein the antibody is a humanizedantibody.
 79. The antibody according to any one of claims 63-77, whereinthe antibody is a chimeric antibody.
 80. A composition comprising anisolated single chain antibody that specifically binds to mature humanTNF-α, wherein when the antibody binds mature human TNF-α, the inductionof endothelial procoagulant activity is inhibited, wherein the isolatedantibody competitively inhibits a second antibody or fragment thereoffor specific binding to mature human TNF-α; and wherein the secondantibody is MAb 42 (ECACC Accession No. 89080304).
 81. The compositionaccording to claim 80, wherein the antibody inhibits the induction oftumor fibrin deposition activity.
 82. The composition according to claim80, wherein the antibody has no effect on tumor receptor binding toTNF-α.
 83. The composition according to claim 82, wherein the antibodyhas no effect on cytotoxicity.
 84. The composition according to claim80, wherein the antibody has no effect on cytotoxicity.
 85. Thecomposition according to claim 84, wherein the antibody has no effect ontumor regression.
 86. The composition according to claim 83, wherein theantibody has no effect on tumor regression.
 87. The compositionaccording to claim 82, wherein the antibody has no effect on tumorregression.
 88. The composition according to claim 80, wherein theantibody has no effect on tumor regression.
 89. The compositionaccording to claim 70, where comparative binding specificity between theantibody and MAb 42 (ECACC Accession No. 89080304) is determined byantibody-antibody competition assays in the presence of mature humanTNF-α.
 90. The composition according to claim 89, wherein the antibodyhas no effect on tumor receptor binding to TNF-α.
 91. The compositionaccording to claim 90, wherein the antibody has no effect oncytotoxicity.
 92. The composition according to claim 89, wherein theantibody has no effect on cytotoxicity.
 93. The composition according toclaim 91, wherein the antibody has no effect on tumor regression. 94.The composition according to claim 89, wherein the antibody inhibitstumor regression.
 95. The composition according to any one of claims80-94, wherein the antibody is a humanized antibody.
 96. The compositionaccording to any one of claims 80-94, wherein the antibody is a chimericantibody.
 97. An isolated single domain antibody or fragment thereofthat specifically binds to mature human TNF-α, wherein when the antibodyor fragment thereof binds mature human TNF-α, the induction ofendothelial procoagulant activity is inhibited, wherein the isolatedantibody or fragment thereof competitively inhibits a second antibody orfragment thereof for specific binding to mature human TNF-α; and,wherein the second antibody is MAb 42 (ECACC Accession No. 89080304).98. The antibody or fragment thereof according to claim 97, wherein theantibody or fragment thereof inhibits the induction of tumor fibrindeposition activity.
 99. The antibody or fragment thereof according toclaim 97, wherein the antibody or fragment thereof has no effect ontumor receptor binding to TNF-α.
 100. The antibody or fragment thereofaccording to claim 99, wherein the antibody or fragment thereof has noeffect on cytotoxicity.
 101. The antibody or fragment thereof accordingto claim 97, wherein the antibody or fragment thereof has no effect oncytotoxicity.
 102. The antibody or fragment thereof according to claim101, wherein the antibody or fragment thereof has no effect on tumorregression.
 103. The antibody or fragment thereof according to claim100, wherein the antibody or fragment thereof has no effect on tumorregression.
 104. The antibody or fragment thereof according to claim 99,wherein the antibody or fragment thereof has no effect on tumorregression.
 105. The antibody or fragment thereof according to claim 97,wherein the antibody or fragment thereof has no effect on tumorregression.
 106. The antibody or fragment thereof according to claim 97,where comparative binding specificity between the antibody or fragmentthereof and MAb 42 (ECACC Accession No. 89080304) is determined byantibody-antibody competition assays in the presence of mature humanTNF-α.
 107. The antibody or fragment thereof according to claim 106,wherein the antibody or fragment thereof has no effect on tumor receptorbinding to TNF-α.
 108. The antibody or fragment thereof according toclaim 107, wherein the antibody or fragment thereof has no effect oncytotoxicity.
 109. The antibody or fragment thereof according to claim106, wherein the antibody or fragment thereof has no effect oncytotoxicity.
 110. The antibody or fragment thereof according to claim108, wherein the antibody or fragment thereof has no effect on tumorregression.
 111. The antibody or fragment thereof according to claim106, wherein the antibody or fragment thereof has no effect on tumorregression.
 112. The antibody or fragment thereof according to any oneof claims 97-111, wherein the antibody is a humanized antibody.
 113. Theantibody or fragment thereof according to any one of claims 97-111,wherein the antibody is a chimeric antibody.
 114. A compositioncomprising an isolated single domain antibody or fragment thereof thatspecifically binds to mature human TNF-α, wherein when the antibody orfragment thereof binds mature human TNF-α, the induction of endothelialprocoagulant activity is inhibited, wherein the antibody or fragmentthereof competitively inhibits a second antibody or fragment thereof forspecific binding to mature human TNF-α; and, wherein the second antibodyis MAb 42 (ECACC Accession No. 89080304).
 115. The composition accordingto claim 114, wherein the antibody or fragment thereof inhibits theinduction of tumor fibrin deposition activity.
 116. THE compositionaccording to claim 114, wherein the antibody or fragment thereof has noeffect on tumor receptor binding to TNF-α.
 117. The compositionaccording to claim 116, wherein the antibody or fragment thereof has noeffect on cytotoxicity.
 118. The composition according to claim 114,wherein the antibody or fragment thereof has no effect on cytotoxicity.119. The composition according to claim 118, wherein the antibody orfragment thereof has no effect on tumor regression.
 120. The compositionaccording to claim 117, wherein the antibody or fragment thereof has noeffect on tumor regression.
 121. The composition according to claim 116,wherein the antibody or fragment thereof has no effect on tumorregression.
 122. The composition according to claim 114, wherein theantibody or fragment thereof has no effect on tumor regression.
 123. Thecomposition according to claim 114, where comparative bindingspecificity between the antibody or fragment thereof and MAb 42 (ECACCAccession No. 90012248) is determined by antibody-antibody competitionassays in the presence of mature human TNF-α.
 124. The compositionaccording to claim 123, wherein the antibody or fragment thereof has noeffect on tumor receptor binding to TNF-α.
 125. The compositionaccording to claim 124, wherein the antibody or fragment thereof has noeffect on cytotoxicity.
 126. The composition according to claim 123,wherein the antibody or fragment thereof has no effect on cytotoxicity.127. The composition according to claim 125, wherein the antibody orfragment thereof has no effect on tumor regression.
 128. The compositionaccording to claim 123, wherein the antibody or fragment thereof has noeffect on tumor regression.
 129. The composition according to any one ofclaims 114-128, wherein the antibody is a humanized antibody.
 130. Thecomposition according to any one of claims 114-128, wherein the antibodyis a chimeric antibody.